Use of three-dimensional microfabricated tissue engineered systems for pharmacologic applications

ABSTRACT

The present invention generally relates to a combination of the fields of tissue engineering, drug discovery and drug development. It more specifically provides new methods and materials for testing the efficacy and safety of experimental drugs, defining the metabolic pathways of experimental drugs and characterizing the properties (e.g., side effects, new uses) of existing drugs. Preferably, evaluation is carried out in three-dimensional tissue-engineered systems, wherein drug toxicity, metabolism, interaction and/or efficacy can be determined.

RELATED APPLICATIONS

This is a continuation-in-part application of International ApplicationSerial Number PCT/US04/01098, filed Jan. 16, 2004, which claims priorityto U.S. Ser. No. 60/440,539, filed on Jan. 16, 2003, the contents eachof which are hereby incorporated herein by reference.

Reference is made herein to U.S. Ser. No. 10/187,247, filed Jun. 28,2002, which claims priority to U.S. Ser. No. 60/367,675, filed Mar. 25,2002, and which is a CIP of 09/560,480, filed Apr. 28, 2000, now U.S.Pat. No. 6,455,311, which claims priority to U.S. Ser. No. 60/165,329,filed Nov. 12, 1999 and to U.S. Ser. No. 60/131,930, filed Apr. 30,1999, the contents each of which are expressly incorporated herein byreference. Reference is also made herein to U.S. Ser. No. 10/038,891,filed Jan. 2, 2002, which claims priority to U.S. Ser. No. 60/259,283filed Jan. 2, 2001. Reference is also made herein to InternationalAppln. No. PCT/US03/29880, which claims priority to U.S. Ser. No.60/412,981, filed on Sep. 23, 2002, and U.S. Ser. No. 60/449,291, filedon Feb. 21, 2003, the contents each of which are hereby incorporatedherein by reference.

Each of the foregoing applications and patents and articles, and eachdocument cited or referenced in each of the foregoing applications andpatents and articles, including during the prosecution of each of theforegoing applications and patents (“application and article citeddocuments”), and any manufacturer's instructions or catalogues for anyproducts cited or mentioned in each of the foregoing applications andpatents and articles and in any of the application and article citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text or in anydocument hereby incorporated into this text, are hereby incorporatedherein by reference. Documents incorporated by reference into this textor any teachings therein can be used in the practice of this invention.Documents incorporated by reference into this text are not admitted tobe prior art. Furthermore, authors or inventors on documentsincorporated by reference into this text are not to be considered to be“another” or “others” as to the present inventive entity and vice versa,especially where one or more authors or inventors on documentsincorporated by reference into this text are an inventor or inventorsnamed in the present inventive entity.

FIELD OF THE INVENTION

The present invention generally relates to a combination of the fieldsof tissue engineering, drug discovery and drug development. It morespecifically provides new methods and materials for testing the efficacyand safety of experimental drugs, defining the metabolic pathways ofexperimental drugs and characterizing the properties (e.g., sideeffects, new uses) of existing drugs. Preferably, evaluation is carriedout in three-dimensional tissue-engineered systems, wherein drugtoxicity, metabolism, interaction and/or efficacy can be determined.

BACKGROUND

Drug discovery and development consists of an arduous testing process,beginning with the demonstration of pharmacological effects inexperimental cell and animal models and ending with drug safety andefficacy studies in patients. It is estimated that only 1 out of 5,000screened compounds receives FDA approval as a safe and effective newmedicine. Approximately 25% of compounds are eliminated in pre-clinicaltoxicological studies. Thus, a significant number of drug candidates inpre-clinical development fail to progress out of this stage due tounacceptable levels of toxicity in test systems.

Multiple pharmacologic parameters are considered when evaluating a drugcandidate. Knowledge of the absorption, distribution, metabolism andexcretion profile (“ADME”) of a drug and its metabolites in humans (andanimals used in toxicology assessments) is crucial to understandingdifferences in effect among individuals in a population and foroptimizing dosimetry. Absorption and bioavailability are standardmeasures of the amount of biologically active material distributed tothe systemic circulation or local site of action. Duration of drugaction is often dependent on how rapidly the body eliminates the activemolecules, either through metabolism, which involves chemicalmodification by drug-metabolizing enzymes, or by excretion, whichinvolves binding and transport away from biologically active sites inthe body. Thus, typical pre-clinical studies involve monitoringpermeation across epithelial membranes (e.g., gastrointestinal mucosa),studies of drug metabolism, identification of plasma protein binding andevaluation of transport into and out of tissues, especially organs thateliminate drug products, such as the kidney and liver.

New Drug Applications, or NDAs, are submitted on the basis of dataobtained from a small number of patients (>10,000), which is usually notindicative of the general population at large. Often, limited toxicityis observed and the selected patients have relatively normal organfunction. Toxicity refers to any unwanted effect on normal structural orfunctional integrity. A toxic dose refers to a dose producing anunwanted or overly exaggerated pharmacological effect in a subject(i.e., the dose received by a subject when the first truly toxic signsdevelop). Failure in development of candidate drugs often occurs when anunacceptable level of toxicity develops.

Often, healthy subjects take part in early stage clinical studies.However, patient responses to drugs are typically more complex and lesspredictable than responses in the healthy subjects. The chances ofadverse drug effects in patients are greatly increased due to increasedsusceptibility (e.g., increased susceptibility due to drug to druginteractions and comorbid conditions). There can be significantdifferences in toxicology and metabolism among groups of patients thatare not detected until the after drug has advanced from pre-clinicalstudies. In the worst case, significant effects on a patient populationare not detected until after a drug receives approval from the Food andDrug Administration. Therefore, pre-clinical approaches to identifytoxicity in the early phases of drug discovery represent an importantstep towards efficient drug development.

Current pre-clinical toxicity and pharmacology studies utilize in vitroassays involving cultured cells or subcellular organelles, as well as invivo animal models to investigate drug metabolism, toxicity and possibleefficacy. While technological advances in cell, molecular, andbiochemical assays have made significant strides, a number of problemsstill exist. First, in vitro assays using purified or recombinantenzymes and cell cultures provide the first step in determiningpharmacologic and toxicologic parameters to be used thereafter in animalmodels, but are often too simplistic to account for the multifactorialevents that occur during drug metabolism in a human system or humanorgan. Second, data obtained in animal models cannot be reliablyextrapolated to human systems. Third, many drugs used to treat chronicdiseases such as HIV infection or Alzheimer's disease necessitate dosingregimens that are applied over long periods of time, and in some cases,over the lifetime of an individual. Currently, development of chronictoxicity is most practically observed during long-term patient use.

The high attrition rate of drug candidates is a major economic deterrentin the pharmaceutical industry, as drug failure may be identified onlyafter great time and expense are invested. These failures can beattributed, in part, to a lack of effective pre-clinical models andassay systems. Accordingly, there is a great need in the art to developan in vitro human system that can effectively evaluate the pharmacologicand toxicologic properties of drug candidates. Improved in vitro modelsystems will allow the drug development process to reliably predict thein vivo response before the drug reaches the clinic, decreasing time,expense and significant risks to patient health. In addition, manyserious diseases (e.g., hepatitis C) lack reliable animal model systems,a problem that has severely handicapped the drug discovery process.Improved in vitro model systems will also provide an opportunity formeaningful pre-clinical experimentation, which is essential for thedevelopment of therapeutics in the absence of animal model systems.

OBJECTS AND SUMMARY OF THE INVENTION

The invention provides a method for determining metabolism of a testagent in a tissue, the method comprising:

-   -   A) providing a test agent to a three-dimensional tissue        engineered system;    -   B) incubating the test agent in the presence of an enzyme within        said system, such that an enzyme-substrate complex is formed        between the enzyme and the test agent; and    -   C) detecting one or more metabolites of the test agent.

Three-dimensional tissue-engineered systems of the invention cancomprise liver tissue, kidney tissue, cardiac tissue, cartilage tissue,or bone marrow tissue, and combinations thereof. Preferably, thethree-dimensional tissue engineered system of the invention comprisesmicrofabricated polymer scaffolds.

Test agents include, but are not limited to, opioid analgesics,anti-inflammatory drugs such as antihistamines and non-steroidalanti-inflammatory drugs (NSAIDs), diuretics such as carbonic anhydraseinhibitors, loop diuretics, high-ceiling diuretics, thiazide andthiazide-like agents, and potassium-sparing diuretics, agents thatimpinge on the renal and cardiovascular systems such as angiotensinconverting enzyme inhibitors, cardiac drugs such as organic nitrates,calcium channel blockers, sympatholytic agents, vasodilators,β-adrenergic receptor agonists and antagonists, α-adrenergic receptoragonists and antagonists, cardiac glycosides, anti-arrhythmic drugs,agents that affect hyperlipoproteinemias such as3-hydroxymethylglutaryl-coenzyme A (HMG-CoA) inhibitors, anti-neoplasticagents such as alkylating agents, antimetabolites, natural products,antibiotics, and other drugs, immunomodulators, anti-diabetic agents,and anti-microbial agents such as antibacterial agents, antiviralagents, antifungal agents, antiprotozoal agents, and antihelminthicagents.

Enzyme-substrate complexes can comprise enzymes including, but notlimited to, cytochrome P450, alkaline phosphatase, α-galactosidase,β-galactosidase, α-glucosidase, β-glucosidase, α-glucuronidase,β-glucuronidase, α-amylase, NADPH-cytochrome P450 reductase, cytochromeb₅, N-demethylase, O-demethylase, acetylcholinesterase,pseudocholinesterase, epoxide hydrolase, amidases, uridine diphosphate(UDP)-glucuronosyltransferases, phenol sulfotransferase, alcoholsulfotransferase, sterid sulfotransferase, and arylaminesulfotransferase, UDP-glycosyltransferases, purinephosphoribosyltransferase, N-acetyltransferases, glutathioneS-transferase, phenylethanolamine N-methyltransferase, non-specificN-methyltransferase, imidazole N-methyltransferase,catechol-O-methyltransferase, hydroxyindole-O-methyltransferase,S-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase,xanthine oxidase, monoamine oxidases, diamine oxidases, flavoproteinN-oxidases, hydroxylases, aromatases, cysteine conjugate β-lyase, andalkylhydrazine oxidase. The enzyme can be endogenously expressed in thetissue, and can have either normal enzymatic activity or alteredenzymatic activity, for example, such as where the enzyme contains apolymorphism or mutation.

The enzyme can be a recombinant enzyme.

In a preferred embodiment, the enzyme is cytochrome P450.

Enzyme metabolites can be detected by methods including, but not limitedto, liquid chromatography, mass spectrometry, nuclear magneticresonance, or spectrophotometry.

The invention provides a method for determining toxicity of a test agentin a tissue, the method comprising:

-   -   A) providing a test agent to a three-dimensional tissue        engineered system;    -   B) incubating the test agent in the presence of the tissue; and    -   C) detecting an undesired effect.        The test agent can be provided for any duration selected by one        of skill in the art, but preferably, the test agent is provided        for at least 24 hours. Methods of the invention are also well        suited for providing the test agent for longer durations (e.g.,        90 days or longer).

The undesired effect can comprises carcinogenicity, cell death, changesin gene expression, changes in protein expression or irregularmetabolism and combinations thereof. Other undesired effects can bereadily identified by one skilled in the art, and may be specific to thetype of test agent provided, or the type of tissue that the test agentis provided to.

Carcinogenicity can be detected by changes in gene expression, changesin protein levels, abnormal cell proliferation, or changes in expressionof antigenic determinants and combinations thereof.

Cell death can be detected by vital dyes, lactate dehydrogenase release,caspase activity, annexin V staining, phosphatidylserine staining orTUNEL assay.

Changes in gene expression can be detected by microchip analysis,RT-PCR, in situ hybridization, fluorescence in situ hybridization orNorthern analysis.

Changes in protein expression can be detected by quantitative Westernblot, immunohistochemistry, immunofluorescence, enzyme-linkedimmunosorbent assay, amino acid sequence analysis, fluorescenceactivated cell sorting or protein concentration assays.

Irregular metabolism can be indicated by detecting abnormal enzymefunction in enzymes including, but not limited to, cytochrome P450,alkaline phosphatase, α-galactosidase, β-galactosidase, α-glucosidase,β-glucosidase, α-glucuronidase, β-glucuronidase, α-amylase,NADPH-cytochrome P450 reductase, cytochrome b₅, N-demethylase,O-demethylase, acetylcholinesterase, pseudocholinesterase, epoxidehydrolase, amidases, uridine diphosphate (UDP)-glucuronosyltransferases,phenol sulfotransferase, alcohol sulfotransferase, steridsulfotransferase, and arylamine sulfotransferase,UDP-glycosyltransferases, purine phosphoribosyltransferase,N-acetyltransferases, glutathione S-transferase, phenylethanolamineN-methyltransferase, non-specific N-methyltransferase, imidazoleN-methyltransferase, catechol-O-methyltransferase,hydroxyindole-O-methyltransferase, S-methyltransferase, alcoholdehydrogenase, aldehyde dehydrogenase, xanthine oxidase, monoamineoxidases, diamine oxidases, flavoprotein N-oxidases, hydroxylases,aromatases, cysteine conjugate β-lyase, and alkylhydrazine oxidase.

In one embodiment, methods of the invention are used to identify testagents having anti-viral activity against hepatitis C. Accordingly, theinvention provides a method for determining efficacy of a test agent,wherein efficacy comprises activity sufficient to decrease or eliminatehepatitis C virus in liver tissue, the method comprising:

-   -   A) providing a test agent to a three-dimensional liver tissue        engineered system;    -   B) incubating the test agent in the presence of the tissue; and    -   C) measuring levels of hepatitis C virus.

The invention further provides a method for determining efficacy of atest agent, wherein efficacy comprises activity sufficient to decreaseor eliminate hepatitis C virus in liver tissue, the method comprising:

-   -   A) providing a test agent to a three-dimensional liver tissue        engineered system;    -   B) incubating the test agent in the presence of the tissue; and    -   C) detecting improved liver function.        Improved liver function is indicated by detecting normal enzyme        levels, histology or protein production and combinations        thereof.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the invention to specific embodiments described, maybe understood in conjunction with the accompanying Figures, incorporatedherein by reference, in which:

FIG. 1 shows a schematic describing a process for the production of acomplex structure comprising channels wherein all channels do not havethe same depth. The steps of the process are as follows: The processbegins with a substrate wafer (A); Masking material is deposited (B);Masking material is patterned (C); Substrate wafer is etched (D); asecond masking layer is deposited (E); The second masking layer ispatterned (F); The substrate wafer is etched again (G); The maskinglayer is removed (H).

FIG. 2 shows a schematic of a pattern etched using aninductively-coupled plasma (ICP) system.

FIG. 3A shows a silicon wafer with a network of microchannels and FIG.3B shows a polymer scaffold with a network of microchannels.

FIG. 4 shows a schematic of an etched surface showing a branchingstructure that branches out from a single inlet and then converges backinto a single outlet.

FIGS. 5 A, B, and C show schematics of a cross-sectional view ofdifferent etched channels in the surface of FIG. 4.

FIG. 6 shows schematic diagram of a cross section of an apparatus fortissue engineering and artificial organ support. The apparatus in FIG.6A comprises a compartment for circulatory flow (1), a semi-permeablemembrane for mass transfer of oxygen, nutrients and waste (2), and acompartment for functional cells and excretory system. FIG. 6B shows theapparatus of 6A seeded with vascular cells or cells that form lumen(e.g. biliary ducts) (4) and functional cells (e.g. hepatocytes) (5).

FIG. 7 shows electron micrographs of a semi-permeable membrane madeusing the TIPS procedure.

FIG. 8 shows a schematic top drawing of a mold or polymer scaffold. Thetriangles represent areas coated with cell adhesion molecules to promotethe adhesion of cells (e.g. hepatocytes). The white areas between thetriangles represent microchannels; in some applications, they are notcoated with cell adhesion molecules, and so are open for colonization bycells that can form vascular tissue (e.g. endothelial cells). The blackcircle in the middle of each hexagon is a vertical through-hole.

FIG. 9 shows how the organ of FIG. 6 can be connected to a fluid byanastomosis of the inlet and outlet.

FIG. 10 shows a set of bar graphs demonstrating continued albuminproduction by hepatocyte cells cultured in a polymer scaffold of theinvention. Albumin concentration in culture medium was measured every 24hours for 5 days pre-cell detachment using an enzyme linkedimmunosorbent assay. No significant differences were observed betweenday 2, day 3, and day 4 (p<0.05 by the paired t-test).

FIG. 11A shows a sample vascular branching network pattern used forsilicon and pyrex wafer micromachining. FIG. 11B shows the opticalmicrograph or portion of a capillary network etched into a siliconwafer. FIG. 11C shows a scanning electron micrograph of an anisotrophicetching process used to form angled sidewall trenches.

FIG. 12 shows phase-contrast photographs of small hepatocytes andnonparenchymal cells cultured on regular culture flasks. FIG. 12A showscells in culture at Day 3. FIG. 12B shows cells in culture at Day 5.FIG. 12C shows cells in culture at Day 10. Scale bar, 100 μm (originalmagnification ×100).

FIG. 13 shows a cell sheet lifted from a silicon wafer. FIG. 13A showsmacroscopic appearance and FIG. 13B shows microscopic appearance(original magnification ×30).

FIG. 14 shows albumin production by small hepatocytes at day 3, 5, 7,and 10 (μg/day).

FIG. 15 shows H & E staining of implanted constructs. FIG. 15A showsconstructs at 2 weeks. Arrows indicate bile ductular structures. FIG.15B shows constructs at 1 month. Arrows indicate bile ductularstructures. FIG. 15C shows constructs at 2 months. The large clusters ofhepatocytes over five cell layers thick were observed at 1 and 2 months.FIG. 15D shows constructs at 1 month. The implanted construct wasoccupied by the bile ductular structures.

FIG. 16 shows immunohistochemical staining of implanted constructs. FIG.16A shows pan-cytokeratin staining at 1 month. FIG. 16B shows albuminstaining at 1 month. Arrows indicate bile ductular structures. FIG. 16Cshows transferrin staining at 1 month. Arrows indicate bile ductularstructures. FIG. 16D shows GGT staining at 1 month. Arrows indicate bileductular structures with luminal staining. Arrow heads indicate slightlystained hepatocytes.

FIG. 17 shows H & E staining at 1 month. Arrow indicates the bileductular structure composed of both biliary epithelial cells and ahepatocyte. Arrow heads indicate the bile ductular structures composedof biliary epithelial cells.

FIG. 18 shows transmission electron microscopy (TEM) of an implantedconstruct. (A) Low magnification (×2500). (B) High magnification(×15000).

FIG. 19 shows the area occupied by implanted constructs (μg²/section).Total area and bile ducts area are expressed as mean±SD.

FIG. 20 shows a schematic diagram of a micromachined apparatus fortissue engineered renal replacement. The apparatus comprises acompartment with a glomerular endothelial filter for circulatory flow(42), a semi-permeable membrane for mass transfer of oxygen, nutrientsand waste (44), and a compartment with a proximal tubule networkexcretory system, which includes inlets for filtration of urine (46).

FIG. 21 shows a cross section of a micromachined apparatus for tissueengineered renal replacement. The apparatus comprises a compartment witha glomerular endothelial filter for circulatory flow, a semi-permeablemembrane for mass transfer of oxygen, nutrients and waste, and acompartment with a proximal tubule network excretory system, whichincludes inlets for filtration of urine. Each compartmentalized layer ofthe apparatus comprises a biocompatible polymer and the layers areseparated by a semi-permeable membrane comprising a microporous polymer.

FIG. 22 shows a cross section of a micromachined apparatus for tissueengineered renal replacement. The direction of flow of glomerularultrafiltrate is shown. Flow originates in the layer comprisingglomerular endothelium, passes through the semi-permeable membrane tolayer comprising the proximal tubule network where reabsorption occurs.

FIG. 23 shows a cross section of a micromachined apparatus for tissueengineered renal replacement comprising multiple stacked layers. Theapparatus comprises repeating, stacked units, each unit comprising acompartment with a glomerular endothelial filter for circulatory flow, asemi-permeable membrane for mass transfer of oxygen, nutrients andwaste, and a compartment with a proximal tubule network excretorysystem, which includes inlets for filtration of urine.

FIG. 24 shows human microvascular cells at 14 days after seeding inmicrochannels.

FIG. 25 shows proximal tubule cells growing in a poly dimethyl-siloxane(PDMS) polymer scaffold at approximately 5 hours after seeding.

FIG. 26 shows proximal tubule cells growing in a poly dimethyl-siloxane(PDMS) polymer scaffold at 2 days after seeding.

FIG. 27 shows proximal tubule cells growing in a poly dimethyl-siloxane(PDMS) polymer scaffold at 6 days after seeding.

FIG. 28 shows a three-dimensional network having a very large number ofvertical interconnects between layers. FIG. 42 depicts a full version ofthe design as shown in FIG. 28.

FIG. 29 is a diagram illustrating a system for designing and modelingfluidic networks.

FIG. 30 illustrates a network, referred to as Testnet-0.

FIG. 31 is a diagram showing capillary bed topology for a network,Testnet-0, created using node-vessel data.

FIG. 32 is a diagram illustrating the topology of a network, referred toas Testnet-1.

FIG. 33 illustrates a branching sequence in which a fractal algorithmwas used to create a hexagonal pattern. In FIG. 33A, the pattern beginsby branching in three directions from a single node. In FIG. 33B, thesame branching pattern is applied to nodes other than the starting node.FIG. 33C shows the hexagonal pattern created by the fractal. FIG. 33Dshows that the pattern can be allowed to grow to any size.

FIG. 34 illustrates a three-dimensional design, Hextak, in accordancewith an embodiment of the invention. The layers in Hextak containvessels in the plane of the wafer. Black lines mark vessels in the planeof the wafer; red and blue circles mark locations where vertical vesselsmeet these patterns. FIG. 34A shows layer B; FIG. 34B shows layer D.

FIG. 35 illustrates four separate layers of vertical vessels in theHextak design. FIG. 35A shows layer A; FIG. 35B shows layer C; FIG. 35Cshows layer E; and FIG. 35D shows layer F.

FIG. 36 is a diagram showing the schematics of the completedtwo-dimensional network designs for Testnet-0 (FIG. 36A) and Testnet-1(FIG. 36B).

FIG. 37 is a diagram of a section of Hextak where each vessel is plottedas an individual three-dimensional element.

FIG. 38 shows fluidic resistance of vessels with varying widths (FIG.38A) and varying lengths (FIG. 38B).

FIG. 39 is a diagram showing a comparison of the prediction made by thenetwork modeling software to experimental data for the total flow ratethrough the vessel versus the pressure drop across the device for aTestnet-1.

FIG. 40 shows a two-dimensional pattern produced by a fractal algorithmin accordance with an embodiment of the invention.

FIG. 41 shows a sample initial network in accordance with an embodimentof the invention.

FIG. 42 shows a full network appropriate for use as support for a tissueengineered organ.

FIG. 43 a depicts a single-pass flow microfabricated device set up. FIG.43 b depicts microfabricated devices with Hep G2/C3a connected tosyringe pumps.

FIG. 44 a depicts a picture of a microfabricated device containing HepG2/C3 cells. FIG. 44 b depicts a picture of Hep G2/C3a cells in tissueculture flask stained with Live/Dead assay demonstrating appearance ofhealthy Hep G2/C3a under normal culture conditions.

FIG. 45 depicts Hep G2/C3a cells in a microfabricated device after 2weeks incubation and stained with Live/Dead stain (Molecular Probes).

FIG. 46 is a graph demonstrating the amount of ECOD and its subsequentbreakdown products in picomoles per cell after 1, 3, 5, 7, and 10 daysof incubation in the microfabricated device.

A related graph shown in FIG. 47 depicts the same data in terms ofmicromoles of ECOD or its metabolites

FIGS. 48-51 depict graphs showing each individual metabolite alone onthe same time scale.

DETAILED DESCRIPTION Definitions

As used herein, the term “toxicity” is defined as any unwanted effect onhuman cells or tissue caused by a test agent, or test agent used incombination with other pharmaceuticals, including unwanted or overlyexaggerated pharmacological effects. An analogous term used in thiscontext is “adverse reaction.”

In the pharmaceutical arts, the term “efficacy” can describe thestrength of a response in a tissue produced from a single drug-receptorcomplex. In the context of this disclosure, “efficacy” can also bedefined as a response elicited by a drug or test agent that improves thephenotype of a cell or tissue.

A “test agent” is any substance that is evaluated for its ability todiagnose, cure, mitigate, treat, or prevent disease in a subject, or isintended to alter the structure or function of the body of a subject. Atest agent in an embodiment can be a “drug” as that term is definedunder the Food Drug and Cosmetic Act, §321(g)(1). Test agents include,but are not limited to, chemical compounds, biologic agents, proteins,peptides, nucleic acids, lipids, polysaccharides, supplements,diagnostic agents and immune modulators.

“Pharmacokinetics” refers to the actions of the body on a drug.Pharmacokinetic processes include, but are not limited to, absorption,distribution, metabolism, and elimination of drugs.

“Pharmacodynamics” refers to the actions of a drug on the body. Becausecertain classes of drugs exhibit similar effects on the body,pharmacodynamic properties determine the group in which a drug or agentis classified.

An “agonist” is a drug, agent, or compound that binds to and activatesits cognate receptor in some fashion, which directly or indirectlybrings about a physiological effect.

An “antagonist” is an agent that binds to a receptor, and which in turnprevents binding by other molecules.

“Phase I metabolism” refers to biochemical reactions that usuallyconvert the parent drug, agent, or compound by introducing or unmaskinga functional group, including but not limited to, hydroxyl, amino, orsulfhydryl groups. The products of Phase I metabolism are ofteninactive, though in some instances activity is only modified or evenhigher than the parent drug.

“Phase II metabolism” encompasses biochemical reactions that couple orconjugate polar molecules to parent drugs or their phase I metabolitesthat contain suitable functional groups for conjugation. Phase IImetabolic reactions require energy. Phase II metabolism can occur beforeor in the absence of Phase I reactions.

A “substrate” is a molecule that binds to the active site of the enzyme,and upon which an enzymatic reaction can be catalyzed. As used herein, a“substrate” is typically the test agent, or a metabolite thereof, whichacts as a substrate for one or more metabolic enzymes.

“Comprises,” “comprising,” “containing” and “having” and the like canhave the meaning ascribed to them in U.S. patent law and can mean“includes,” “including,” and the like; “consisting essentially of” or“consists essentially” likewise has the meaning ascribed in U.S. patentlaw and the term is open-ended, allowing for the presence of more thanthat which is recited so long as basic or novel characteristics of thatwhich is recited is not changed by the presence of more than that whichis recited, but excludes prior art embodiments.

Methods of the Invention

Three-dimensional tissue engineered systems of the invention are usefulfor studying several parameters of a test agent, including metabolism,toxicity and efficacy. Methods of the invention can be used to screenexperimental drugs or “test agents” that have no known metabolic orpharmacokinetic profile, in order to obtain such information, includinginformation necessary to assess toxicity. Toxicity can often occur as aresult of drug-to-drug interactions. Thus, methods of the invention canbe used to study the combination of test agents with known drugs orother test agents. These methods are particularly relevant to use inclinical settings since many patients are treated with multiple drugs.

In general, test agents are incubated with the three-dimensional tissueengineered systems of the invention in a dosage range estimated to betherapeutic and for a duration sufficient to produce an effect (e.g.,metabolic effects or effects indicating to toxicity or efficacy). Theincubation time can range between about 1 hour to 24 hours, or can beextended as necessary for several days or even weeks. The incubationconditions typically involve standard culture conditions known in theart, including culture temperatures of about 37° C., and culture mediumscompatible with the particular cell type selected.

Test agents that can be analyzed according to methods of the inventioninclude, but are not limited to, opioid analgesics, anti-inflammatorydrugs such as antihistamines and non-steroidal anti-inflammatory drugs(NSAIDs), diuretics such as carbonic anhydrase inhibitors, loopdiuretics, high-ceiling diuretics, thiazide and thiazide-like agents,and potassium-sparing diuretics, agents that impinge on the renal andcardiovascular systems such as angiotensin converting enzyme (ACE)inhibitors, cardiac drugs such as organic nitrates, calcium channelblockers, sympatholytic agents, vasodilators, β-adrenergic receptoragonists and antagonists, α-adrenergic receptor agonists andantagonists, cardiac glycosides, anti-arrhythmic drugs, agents thataffect hyperlipoproteinemias such as 3-hydroxymethylglutaryl-coenzyme A(HMG-CoA) inhibitors, anti-neoplastic agents such as alkylating agents,antimetabolites, natural products, antibiotics, and other drugs,immunomodulators, anti-diabetic agents, and anti-microbial agents suchas antibacterial agents, antiviral agents, antifungal agents,antiprotozoal agents, and antihelminthic agents, but are not limited tothese agents.

Metabolism

The cytochrome P450 enzyme family is the major catalyst of drugbiotransformation reactions, and can be used in vitro to determine drugbinding and drug metabolism in the tissue-engineered systems of thepresent invention. Assays for monitoring enzyme metabolism, includingcytochrome P450 enzyme function, can be performed according to methodswell known in the art. Several of these assays are described below.

The cytochrome P450 superfamily of enzymes, which are primarily liverenzymes, catalyzes a wide variety of oxidative and reductive reactionsand has activity towards a chemically diverse group of substrates.Cytochrome P450 enzymes are heme-containing membrane proteins localizedin the smooth endoplasmic reticulum of numerous tissues. Thesehemoproteins are in close association with a second membrane protein,NADPH-cytochrome P450 reductase. Oxidative biotransformations catalyzedby cytochrome P450 monooxygenases include aromatic and side chainhydroxylation, N-, O-, and S-dealkylation, N-oxidation, sulfoxidation,N-hydroxylation, deamination, dehalogenation, and desulfuration.Cytochrome P450 enzymes, generally under conditions of low oxygentension, also catalyze a number of reductive reactions. The only commonstructural feature of the diverse group of drugs oxidized by cytochromeP450 enzymes is their high lipid solubility.

A plurality of cytochrome P450 gene families has been identified inhumans, and a number of distinct cytochrome P450 enzymes often existwithin a single cell. The cytochrome P450 multigene family is classifiedby sequence similarity of the individual proteins. A given cytochromeP450 family is further divided into subfamilies, such that proteinsequences within the same subfamily are >55% identical. The cytochromeP450 1, 2, 3, and 4 families (CYP1, CYP2, CYP3, CYP4) encode the enzymesinvolved in the majority of all drug biotransformations, while the geneproducts of the remaining cytochrome P450 families are important in themetabolism of endogenous compounds, such as steroids and fatty acids.The relevant CYP enzymes that are expressed in humans include, but arenot limited to, CYP1A1, CYP1A2, CYP2A3, CYP2B6, CYP2B7, CYP2B8, CYP2C8,CYP2C9, CYP2C10, CYP2D6, CYP2D7, CYP2D8, CYP2E1, CYP2F1, CYP3A3, CYP3A4,CYP3A5, and CYP4B1. As a result of the relatively low substratespecificity among the cytochrome P450 proteins, two or more individualenzymes often can catalyze a given biotransformation reaction. CYP3A4 isinvolved in the biotransformation of a majority of all drugs and isexpressed at significant level extrahepatically.

Cytochrome P450 is a hemoprotein that when reduced and complexed withcarbon monoxide, a characteristic absorption spectrum results. Thereduced carbon monoxide spectrum of cytochrome P450 absorbs maximally ataround 450 nm and the extinction coefficient for the wavelength couple450-490 nm has been accurately determined to be 91 mM⁻¹ cm⁻¹, thusallowing quantitative determination of this hemoprotein. If highturbidity is present in the sample containing cytochrome P450,spectrophotometric determination of the hemoprotein can be carried outin a split beam instrument, i.e. one containing both a sample andreference compartment to offset turbidity. Solid sodium dithionite, forexample, is used as a reducing agent, and the samples can be gassed withcarbon monoxide shortly after dithionite addition (the reduced ferrousform of CYP450 is relatively unstable). Excessively high gas flow ratescan result in frothing and protein denaturation. If a prominent peak isobserved at 420 nm after gassing with carbon monoxide, this isindicative of the presence of inactive cytochrome P420, and is to beavoided.

The tissue content of cytochrome b₅ can also be analysed using the samesample. If both cytochrome P450 and cytochrome b₅ concentration arerequired from the same sample, the cytochrome b₅ must be determinedfirst as in the method given below. This is achieved by determining thedifference absorbance spectrum of NADH-reduced versus oxidizedcytochrome b₅. The reduced, ferrous form of cytochrome b₅ has anabsorbance maximum at 424 nm in difference spectrum and the extinctioncoefficient for the wavelength couple 424-490 nm is 112 mM⁻¹ cm⁻¹. NADHcan be used as the reductant because of the presence of the flavoproteinenzyme NADH-cytochrome b₅ reductase in tissue preparations, an enzymethat relatively specifically and quantitatively reduces cytochrome b₅.

Many agents can bind to cytochrome P450, resulting in characteristicperturbations of the absorbance of the heme iron. The absorbance changescan be utilized to quantitatively describe drug binding to thehemoprotein, resulting in the determination of the apparent spectraldissociation constant (K_(s)) and maximum spectral change elicited bythe drug (ΔA_(max)). These two parameters are formally similar to theK_(m) and V_(max) values described by Michaelis-Menten kinetics forenzyme-catalysed reactions. In the broadest sense, K_(s) is a measure ofdrug affinity for cytochrome P450 and ΔA_(max) is the maximum spectralchange. These two spectral parameters are therefore of use in comparingthe interactions of test agents with various forms of cytochrome P450 orin comparing the interactions of different test agents, or combinationsthereof, with the same form of cytochrome P450.

NADPH-cytochrome c (P450) reductase is a flavoprotein enzyme localizedin the microsomal fraction of the liver that transfers the necessaryreducing equivalents from NADPH to cytochrome P450 during certain drugmetabolism reactions as:

NADPH_(→)NADPH-cytochrome c(P450)reductase→cytochrome(P450)

As the reduction of cytochrome P450 is relatively difficult to assaydirectly, a simplified determination of enzyme activity is widely used,utilizing exogenous cytochrome c (oxidized, ferric form) as anartificial election acceptor. Accordingly, the reduction of cytochrome cby NADPH-cytochrome c (P450) reductase mirrors the reduction ofcytochrome P450. The principle of the method is that oxidized (ferric)cytochrome c has a characteristic absorption spectrum, as does thereduced (ferrous) form. However, the reduced form has a characteristicabsorption band at 550 nm, a band that is absent in the oxidized form.Therefore, the enzyme activity can be conveniently assayed by measuringthe increase in absorbance at 550 nm as a function of time.

Many drugs are hydroxylated in the liver by the cytochromeP450-dependent, mixed-function oxidase system, and the 4-hydroxylationof aniline is a convenient, reproducible assessment of this reaction as:

The 4-aminophenol metabolite produced is chemically converted to aphenolindophenol complex with an absorption maximum at 630 nm and isbased on the method of Schenkman et al. Addition of aniline HCl solutioninitiates the enzyme reaction. The reaction is terminated with ice-cold20% trichloroacetic acid and centrifuged to yield a clear solution (5min in a bench centrifuge at maximum speed is usually sufficient). Thesupernatant can then be added to a 1% phenol solution in a separate testtube in the presence of sodium carbonate. After a 30-minute incubation,the absorbance is read at 630 nm.

N-demethylation of drugs is a common metabolic pathway and usuallyproceeds by initial hydroxylation at the α-carbon atom and subsequentbreakdown of the carbinolamine intermediate liberating formaldehyde.Therefore, if the formaldehyde produced could be measured, this wouldthen yield an appropriate assay for the N-demethylase activity.Formaldehyde may be trapped in solution as the semicarbazone andmeasured by the colorimetric procedure of Nash (1953), based on Hantzschreaction. A solution including semicarbazide, MgCl₂, and aminopyrine canbe added to microsomes or post-mitochondrial supernatant, and thereaction occurs over 30 minutes. The reaction is terminated by additionof zinc sulfate on ice. A saturated barium hydroxide solution is addedto the mix, and centrifuged to a clear supernatant. The Nash reagent isthen added to the supernatant and incubated at 60° C. for 30 minutes.After cooling the tubes, the absorbance is read at 415 nm.

In a similar manner to N-demethylation, many drugs can undergoO-demethylation reactions, catalyzed by the microsomal, cytochromeP450-dependent, mixed-function oxidase system. A useful substrate tomonitor O-demethylation reactions is 4-nitroanisole, which is convertedto 4-nitrophenol as

The 4-nitrophenol thus produced, forms an intense yellow color at pH 10,with an absorbance maximum at 400 nm. Hence the activity of the enzymesystem can be followed spectrophotometrically.

A number of nonspecific esterases and amidases have been identified inthe endoplasmic reticulum of liver, intestine, and other tissues. Suchenzymes include acetylcholinesterase, pseudocholinesterase, otheresterases, epoxide hydrolase, but are not limited to these examples. Thealcohol and amino groups exposed following hydrolysis of esters andamides are suitable substrates for conjugation reactions. Epoxidehydrolase is found in the endoplasmic reticulum of essentially alltissues and is in close proximity to the cytochrome P450 enzymes.Epoxide hydrolase generally is considered a detoxification enzyme;hydrolyzing highly reactive arene oxides generated from cytochrome P450oxidation reactions to inactive, water-soluble transdihydrodiolmetabolites. Proteases and peptidase enzymes are widely distributed inmany tissues and are involved in the biotransformation of polypeptidedrugs.

The glucuronosyl transferase family of enzymes is important in phase IIdrug conjugation reactions. Uridine diphosphate glucuronosyltransferases(UDP-glucuronosyltransferases) catalyze the transfer of an activatedglucuronic acid molecule to aromatic and aliphatic alcohols, carboxylicacids, amines, and free sulfhydryl groups of both exogenous andendogenous compounds to for O-, N-, and S-glucuronide conjugates. TheUDP-glucuronosyltransferases are microsomal enzymes. Their location inthe microsomal membrane facilitates direct access to the metabolitesformed in phase I reactions. In addition to high expression levels inthe liver, UDP-glucuronosyltransferases are also found in the kidney,intestine, brain, and skin.

A useful compound to assess glucuronosyl transferase activity is2-aminophenol, because this phenol readily forms as O-linked glucuronideconjugate in the presence of UDP-glucuronic acid. The assay forglucuronidation of 2-aminophenol is based on the colorimetricdiazotisation method for free primary amino groups. The principle of theanalytical method is based on the observation that when an aqueoussolution of sodium nitrite is added to a cold, acidified solution of anaromatic amine, a diazonium salt is formed. Excess nitrite is removed bythe addition of ammonium sulfamate and the diazonium salt is finallyreacted with a complex aromatic amine (N-naphthylethylene diamine), toproduce a brightly coloured azo compound that can be analysedspectrophotometrically. This method, therefore, detects the amino groupof the 2-aminophenyl glucuronide. The method is relatively specificbecause excess substrate (2-aminophenol) is destroyed under the assayconditions (at pH 2.7) and therefore does not take part in thediazotisation reaction.

As the glucuronosyl transferases usually exhibit enzyme latency in themicrosomal membrane, the assay is carried out in the presence of adetergent (usually Triton X-100) to offset the latency. Ascorbic acid isincluded as an anti-oxidant. The substrate 2-aminophenol can be added totest samples comprising, for example, either microsomal orpost-mitochondrial fraction, at 37° C. in a shaking water bath, and thereaction allowed to proceed for 30 minutes. The reaction is terminatedby addition of ice-cold 20% trichloroacetic acid in phosphate buffer, pH2.7, allowed to stand on ice for 5 minutes and clarified bycentrifugation. Fresh 0.1% sodium nitrite is added, followed by 0.5%ammonium sulfamate, and 0.1% N-naphthylethylene diamine, incubated atroom temperature in the dark for 60 minutes. The absorbance is read at540 nm against the substrate blank.

Sulfation also is an important conjugation reaction for hydroxyl groups.Cytosolic sulfotransferases catalyze the transfer of inorganic sulfurfrom the activated 3′-phosphoadenosine-5′-phosphosulfate donor moleculeto the hydroxyl group on phenols and aliphatic alcohols. Examples ofsulfotransferases include, but are not limited to, phenolsulfotransferase, alcohol sulfotransferase, sterid sulfotransferase, andarylamine sulfotransferase.

UDP-glycosyltransferases transfer glucose moieties in a similar fashionthat glucuronosyltransferases conjugate glucuronic acid to pharmacologicagents. Ribose and deoxyribose sugar moieties can also be added,mediated by enzymes such as purine phosphoribosyltransferase, amongothers.

A family of N-acetyltransferases is responsible for the acetylation ofamines, hydrazines, and sulfonamides. In contrast to most drugconjugates, acetylated metabolites are often less soluble in water thanthe parent drug, a property that prolongs their elimination from thebody. Conjugation of electrophilic metabolites with the tripeptideglutathione represents a major detoxification pathway for drugs andcarcinogens.

The glutathione-S-transferases are a family of isoenzymes that catalysethe conjugation of the endogenous tripeptide glutathione(gamma-glutamylcysteinylglycine) with a large number of structurallydiverse, electrophilic drugs or their metabolites. The glutathioneS-transferase enzymes are expressed in virtually all tissues.Glutathione conjugates are cleaved to cysteine derivatives andsubsequently are acetylated by a series of enzymes located primarily inthe kidney to give N-acetylcysteine conjugates collectively referred toas mercapturic acids. The glutathione-S-transferases consist of twosubunits each of which is inducible by many drugs, and although someexceptions are known, their prime function is in the detoxification ofbiologically reactive electrophiles.

A convenient spectrophotometric method has been developed for theanalysis of glutathione-S-transferase activity based on theenzyme-catalyzed condensation of glutathione with the model substrate2,4-dinitro-1-chlorobenzene. The product formed(2,4-dinitrophenyl-glutathione) absorbs light at 340 nm and theextinction coefficient of this product is known to be 9.6 mM⁻¹ cm⁻¹,thus facilitating the analysis of enzyme activity based on productformation. It known in the art that the glutathione-S-transferaseisoenzymes have similar but overlapping substrate specificities for theelectrophilic substrate to be conjugated. Therefore one substrate thatis readily reactive with a particular isoenzyme may not be substrate foranother isoenzyme. Dinitrochlorobenzene is a good substrate for most ofthe glutathione-S-transferase isoenzymes, when results are interpretedwith the knowledge that observed activity can represent a compositeresult of the activity of each isoenzyme present in the tissuepreparation. One skilled in the art can readily interpret the data toconsider the results as a composite rather than an individual measure ofmetabolic activity.

A glutathione solution can be prepared in the presence ofdinitrochlorobenzene and potassium phosphate buffer, pH 6.5. Since thereaction is measured as a function of time, the reaction is directlyassayed in cuvettes placed in the spectrophotometer. The reaction isinitiated by adding a post-mitochondrial or microsomal fraction fromliver, mixed thoroughly, and the increase in absorbance at 340 nm over a5 minute period should be measured as quickly as possible.

Methylation and conjugation with the amino acids glycine, glutamine, andtaurine are less common reactions for drugs but represent importantreactions for endogenous compounds. Methyltransferases include, but arenot limited to, phenylethanolamine N-methyltransferase, non-specificN-methyltransferase, imidazole N-methyltransferase,catechol-O-methyltransferase, hydroxyindole-O-methyltransferase, andS-methyltransferase.

Other enzymes that are involved in drug metabolism, and that can beassayed in accordance with methods of the invention to determine themetabolic profile of a test agent, include, but are not limited to,alcohol dehydrogenase, aldehyde dehydrogenase, xanthine oxidase, amineoxidases such as monoamine oxidases, diamine oxidases, flavoproteinN-oxidases, and hydroxylases, aromatases, cysteine conjugate β-lyase,α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase,α-glucuronidase, β-glucuronidase, α-amylase, and alkylhydrazine oxidase.

Levels of metabolites, if known, can be detected using methods wellknown in the art as a reflection of metabolic activity, such as liquidchromatography. Liquid chromatography coupled with tandem massspectrometric detection (LC/MS/MS) can be used as an analytical methodto monitor early absorption, distribution, metabolism and eliminationtesting. This method provides excellent sensitivity, specificity andhigh sample throughput. The quantitative selectivity afforded byreaction monitoring on a triple quadrupole instrument precludes the needfor high chromatographic resolution or extensive sample clean up. Usingautomated sample-processing techniques, such as on-line columnswitching, combined with high-sample-density microtiter plates, canfurther maximize analytical throughput. Modern LC/MS/MS also offerslimits of detection extending down to the sub-nanogram per ml rangeusing only minimal quantities of biological matrix.

LC/MS/MS enables rapid and sensitive quantitation of new drugcandidates, as well as providing important structural information onmetabolites. A full scan LC/MS analysis can initially suggest possibleoxidative and/or conjugative metabolic transformations on the basis ofthe ionic species observed. In the MS/MS mode, the instrument can betuned to a selected precursor ion of interest, which is then furtherfragmented to form productions that uniquely identify the metabolic(production scan).

Selectivity can be further enhanced by the quadrupole ion trap, a devicethat “traps” ions in a space bounded by a series of electrodes. Theunique feature of the ion trap is that an MS/MS experiment (or, in fact,multi-step MS experiments) can be performed sequentially in time withina single mass analyzer, yielding a wealth of structural information.Hybrid quadrupole-time-of-flight (Q-TOF) LC/MS/MS systems can also beused for the characterization of metabolite profiles. The configurationof Q-TOF results in high sensitivity in mass resolution and massaccuracy in a variety of scan modes.

Liquid chromatography coupled with nuclear magnetic resonancespectroscopy (LC-NMR) provides a way of confirming absolute molecularconfigurations. A linear ion-trap mass spectrometer possessessignificantly enhanced production-scanning capabilities, while retainingall of the scan functions of a triple quadrupole MS. The ultra-highresolution and sensitivity of Fourier transform ion-cyclotron resonanceMS (FI-ICRMS) can be useful for the analysis and characterization ofbiological mixtures. Data processing and interpretation softwarepackages also enable efficient identification and quantification ofmetabolites using the tissue-engineered devices of the presentinvention.

A widely used method to study in vitro drug metabolism is the use oftissue homogenates. The tissues within the three-dimensional systems ofthe invention can be cultured in the presence of a test agent andharvested to obtain tissue homogenate preparations for use in enzymeanalysis. Preparation of tissue homogenates is well known in the art andinvolves the steps of tissue homogenization and subcellularfractionation to yield two main fractions routinely studied in drugmetabolism: the post-mitochondrial supernatant and the endoplasmicreticulum (microsomal) fraction.

For preparation of the post-mitochondrial supernatant, the tissuehomogenate can be centrifuged as 12,500×g for 15 minutes to pelletintact cells, cell debris, nuclei and mitochondria. The resultantsupernatant (the post-mitochondrial supernatant) is carefully decantedand contains the microsomal plus soluble fractions of the cell.Microsomal tissue fractions can be prepared from the post-mitochondrialsupernatant by one of two centrifugation techniques, one involving theuse of an ultracentrifuge and the other involving a calciumprecipitation of the microsomes at a lower g force.

The ultracentrifugation method uses aliquots (approximately 10-12 ml) ofthe post-mitochondrial supernatant, which are transferred toultracentrifuge tubes and centrifuged at 100,000×g for 45 minutes in arefrigerated ultracentrifuge. After centrifugation, the supernatant isdecanted and discarded and the microsomal pellet resuspended in asuitable buffer containing physiological concentrations of salt, such asTris. This procedure yields the final microsomal suspension.

The calcium precipitation method is based on the calcium dependentaggregation of endoplasmic reticulum fragments and subsequent ‘lowspeed’ centrifugation of the aggregated microsomal particles. Theadvantages of this method are that it is less time-consuming and doesnot require an ultracentrifuge. Aliquots of post-mitochondrialsupernatant are mixed with a final CaCl₂ concentration of 8 mM and leftto stand on ice for 5 min, with occasional gentle swirling.

The mixture is then centrifuged at 27,000×g for 15 min, the supernatantdiscarded and the pellet resuspended by homogenization in a buffer suchas Tris at physiological pH, yielding the microsomal suspension.

The microsomal fractions prepared by both of the above methods may befurther washed by resuspending the microsomal pellet in 0.1 M Trisbuffer, pH 7.4, containing 0.15 M KCl to remove either adventitiousprotein or excess CaCl₂. The microsomal pellet can then precipitated asabove and resuspended in Tris buffer. It is not mandatory to resuspendthe final microsomal preparations in Tris buffer and other buffers suchas phosphate may be used. When comparing tissue fractions for theirability to catalyze drug biotransformation, a measure of the tissueprotein is required. Amongst several methods, protein is readilydetermined by the colorimetric method of Lowry et al. (1951), withreference to a standard curve of bovine serum albumin. The coloredcomplex is a result of a complex between the alkaline copper-phenolreagent used and tyrosine and tryptophan residues of the protein, andcan be detect by spectrophotometer at 705 nm. Other protein detectionmethods are well known in the art and include the Bradford assay.

Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is often anecessary cofactor for many drug biotransformation reactions and servesas a source of reducing equivalents in the reaction (particularlyhydroxylation and demethylation reactions).

Toxicity

There are three general classes of toxicity. Acute toxicity is a toxiceffect that occurs after less than about 24 hours of exposure to thedrug. Subacute toxicity occurs later, after about 14 to 90 days ofexposure to the drug. Chronic toxicity occurs after about 90 days (orlonger) exposure to the drug. Current methods in the art are suboptimalfor use in detecting subacute and chronic toxicity due to therequirement for extended periods of monitoring in a living subject.While methods of the invention can encompass these longer intervals ofexposure, effects may be detected more rapidly, such that the incubationtime for the test agent need not be extended. Accordingly, incubationtimes can range between about 1 hour to 24 hours, or can be extended asnecessary for several days or even weeks.

The undesired effects of toxicity caused by administration of a testagent can be screened in several ways. Tissue engineered systems of theinvention can be used to determine the range of toxic dosimetry of atest agent. The effect of increasing concentrations of the test agent(i.e., dose) on tissues of interest can be monitored to detect toxicity.A toxic effect, when observed, can be equated with a measurement of testagent concentration/cells cm². By calculating the toxic concentrationaccording to the distribution of cells in the tissue engineered system,one of skill in the art can extrapolate to the living system, toestimate toxic doses in subjects of various weights and stages indevelopment.

Using methods of the present invention, various doses of individual testagents and combinations of test agents with other pharmaceuticals willbe screened to detect toxic effects, including but not limited toirregular metabolism, carcinogenicity and cell death. To detectirregular changes in metabolism, standard methods known in the art forassaying metabolite production, including but not limited to glucosemetabolism and enzymatic assays, can be employed. The particularmetabolic pathway assayed, or metabolite measured, can vary according tothe tissue type selected.

In detecting carcinogenicity, cells can be screened for a transformedphenotype using methods well known in the art, for example, methodsdetecting changes in gene expression, protein levels, abnormal cellcycles resulting in proliferation and changes in expression of cellsurface markers, including, but not limited to, antigenic determinants.Gene expression patterns can be determined, for example, by evaluatingmRNA levels of genes of interest according to standard hybridizationtechniques, such as RT-PCR, in situ hybridization, and fluorescence insitu hybridization (FISH), Northern analysis or microchip-basedanalysis. Protein expression patterns can be determined by any methodsknown in the art, for example, by quantitative Western blot,immunohistochemistry, immunofluorescence, and enzyme-linkedimmunosorbent assay (ELISA), amino acid sequence analysis, and/orprotein concentration assays. For details, see Sambrook, Fritsch andManiatis, Molecular Cloning, A Laboratory Manual, 2nd Ed., Cold SpringHarbor Laboratory Press, 1989. Cell counting and/or separationtechniques, such as FACS analysis, can be employed to measureproliferation or detect aberrant cell surface marker expression.

Standard methods well known in the art can also be used to detect celldeath, including but not limited to, tunnel assays. Traditionalapproaches of in vitro toxicology to toxicological screening has been tomeasure comparatively late events in the process of cell death, such aslactate dehydrogenase release or differential counting of viable anddead cells using vital dyes, such as trypan blue,4,6-diaminophenylindole (DAPI), propidium iodide, and LIVE/DEAD® stainavailable from Molecular Probes. Prediction of lethality in vivo is oneproposed application of this type of in vitro screen, although celldeath is not a common mechanism by which the animal's death is inducedfollowing acute exposure to a toxic agent. In contrast, caspaseactivation is at the center the common features of chronic toxicity,cell death, hyperproliferation and inflammatory reactions. Caspaseactivity can be measured relatively quickly after a toxic insult (30 minto 4 hr) by fluorescence spectroscopy, thus lending itself tohigh-throughput screening techniques. Other markers and assays commonlyused to monitor apoptosis or necrosis of cells can include, but are notlimited to, the presence of phosphatidylserine on the outer leaflet ofthe plasma membrane of affected cells, annexin V staining, and terminaldeoxynucleotidyltransferase nick-end labeling assay (TUNEL).

Using methods of the invention, various doses of individual test agentsand combinations of test agents will be screened in panels comprised oftissues having diverse genetic backgrounds to determine thepharmacogenetic toxicity profile of the test agents. For example,multiple doses of, or combinations with, test agents will be screenedfor toxic effects specific to one or more genetic backgrounds. Toxiceffects to be screened for genetic variance include, but are not limitedto, irregular metabolism, carcinogenicity and cell death.

Tissue-engineered devices of the present invention can be modified inparallel to generate a comprehensive array of the currently knowngenetic polymorphisms of different metabolic enzymes. A salient exampleis the CYP450 monooxygenase system, wherein the population comprisesmultiple isoforms and polymorphisms that impinge on and complicatepredictive models of drug metabolism, drug clearance, and toxicity. Forexample, in the metabolism of thiopurines, such as thioguanine, therate-limiting enzyme is a methyltransferase that has differentpolymorphic forms. Polymorphism in the methyltransferases is known toaffect metabolism of the thiopurines. Where the polymorphism gives riseto slower metabolism of the thiopurine, clinical benefit is decreasedand where the polymorphism gives rise to an increased rate ofmetabolism, toxicity can result. Thus, methods of the invention can beused to determine the metabolic profile of various test agents in thepresence of various polymorphic forms of an enzyme, such asmethyltransferase.

In testing for differential toxicity due to polymorphic variation, orother genetic defects, genetically engineered cells comprising geneknockouts or knock-ins of specific enzymes known to affect drugmetabolism and toxicity can be used in the systems of the invention.Cells can be modified using techniques that are known to the skilledartisan, such as RNA interference (RNAi), antisense technology,ribozymes, site-directed mutagenesis, among others.

Efficacy

Efficacy can be detected by measuring individual parameters associatedwith the repair, enhancement, improvement and/or regeneration of adisease model comprising an injured tissue grown in a three-dimensionalsystem of the invention. In disease models of the invention, the injurycan be induced or can be the result of a pre-existing condition in thetissue donor, including conditions relating to inherited geneticabnormalities. Either the induced or pre-existing condition can comprisea weakened state resulting from a previous drug exposure. Test agents,or combinations of test agents, can be analyzed for efficacy in diseasemodels of the invention.

In one embodiment, selected tissues of interest can be treated withagents known in the art to cause cellular damage (e.g., toxins,mutagens, radiation, infectious agents and chemical agents), inducinginjury in the tissue. In another embodiment, selected tissues ofinterest can be altered using standard recombinant techniques to inducea disease state. For example, techniques of homologous recombination canbe used to insert a transgene into a cell, or “knock-out” geneexpression of a gene of interest. For a review of homologousrecombination, see Lewin, B., Genes V, Oxford University Press, NewYork, 1994, pp. 968-997; and Capecchi, M., (1989) Science 244:1288-1292;Capecchi, M., (1989) Trends Genet. 5 (3):70-76. In another embodiment,the selected tissue of interest is injured as a result of an inheritedgenetic defect, which can be a single gene defect or a multifactorialdefect. For a discussion of inherited disorders, see Thompson, McInnesand Willard, Genetics in Medicine, 5^(th) Ed., W.B. Saunders Company,1991.

Tissue engineered systems of the invention can be used to determine therange of effective dosimetry of a test agent. The effect of increasingconcentrations of the test agent (i.e., dose) on tissues of interest canbe monitored to detect efficacy. A therapeutic effect, when observed canbe equated with a measurement of concentration/cells cm². By calculatingthe effective concentration according to the distribution of cells inthe tissue engineered system, one of skill in the art can extrapolate tothe living system, to estimate therapeutic doses in subjects of variousweights.

Using methods of the invention, various doses of individual test agentsand combinations of test agents will be screened in panels comprised oftissues having diverse genetic backgrounds to determine thepharmacogenetic efficacy profile of the test agents. For example,multiple doses of, or combinations with, test agents will be screenedfor efficacy, or the lack thereof, specific to one or more geneticbackgrounds.

Tissues of Interest

Methods of the invention can be carried out using tissues of any kind.The following description provides specific information relating to fivepreferred embodiments of the invention.

1. Liver

A. Toxicity

The liver plays a major role in carbohydrate metabolism by removingglucose from the blood, under the influence of the hormone insulin, andstoring it as glycogen. When the level of glucose in the blood falls,the hormone glucagon causes the liver to break down glycogen and releaseglucose into the blood. The liver also plays an important role inprotein metabolism, primarily through deamination of amino acids, aswell as the conversion of the resulting toxic ammonia into urea, whichcan be excreted by the kidneys. In addition, the liver participates inlipid metabolism by storing triglycerides, breaking down fatty acids,and synthesizing lipoproteins. The liver also secretes bile, which helpsin the digestion of fats, cholesterol, phospholipids, and lipoproteins.

Analysis of metabolic function will indicate toxicity in liver. Thus, inliver tissue engineered systems of the invention, metabolic assays todetect toxicity of a particular test agent are preferred. Metabolicenzymes, including but not limited to, cytochrome P450, alkalinephosphatase, glycolytic enzymes such as α-galactosidase,β-galactosidase, α-glucosidase, β-glucosidase, α-glucuronidase,β-glucuronidase, and α-amylase, NADPH-cytochrome P450 reductase,cytochrome b₅, N-demethylase, O-demethylase, acetylcholinesterase,pseudocholinesterase, among other esterases, epoxide hydrolase,amidases, Uridine diphosphate (UDP)-glucuronosyltransferases, phenolsulfotransferase, alcohol sulfotransferase, sterid sulfotransferase, andarylamine sulfotransferase, UDP-glycosyltransferases, purinephosphoribosyltransferase, N-acetyltransferases, glutathioneS-transferase, phenylethanolamine N-methyltransferase, non-specificN-methyltransferase, imidazole N-methyltransferase,catechol-O-methyltransferase, hydroxyindole-O-methyltransferase, andS-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase,xanthine oxidase, amine oxidases such as monoamine oxidases, diamineoxidases, flavoprotein N-oxidases, and hydroxylases, aromatases,cysteine conjugate β-lyase, and alkylhydrazine oxidase can be tested formetabolic activity using assays well known in the art (this is describedin great detail in other portions of the application). Cytochrome p450enzymes that can be tested include, but are not limited to, CYP1A1,CYP1A2, CYP2A3, CYP2B6, CYP2B7, CYP2B8, CYP2C8, CYP2C9, CYP2C10, CYP2D6,CYP2D7, CYP2D8, CYP2E1, CYP2F1, CYP3A3, CYP3A4, CYP3A5, and CYP4B1.

In a preferred embodiment, the test agent comprises antiviral activity,most preferably, antiviral activity against hepatitis. Currently, thereis a great need for safe and effective treatments for hepatitis(Mutchnick, M. G., et. al., Antiviral Research (1994) 24:245-257). Forexample, clinical tests on the use of the nucleoside analog fialuridine(FIAU) for treatment of chronic hepatitis B were suspended recently dueto drug-related liver failure leading to death in some patients. Testagents demonstrating efficacy against hepatitis can also be screened foracute, subacute and chronic toxicity by monitoring metabolic function,preferably of metabolic function of cytochrome P450 and alkalinephosphatase, following administration.

B. Efficacy

Test agents can be screened for efficacy in tissue engineered systems ofthe invention comprising liver cells affected with diseases including,but not limited to, cancer, diabetes, acute hepatitis, fulminanthepatitis, chronic hepatitis, hepatic cirrhosis, fatty liver, alcoholichepatopathy, drug induced hepatopathy (drug addiction hepatitis),congestive hepatitis, autoimmune hepatitis, primary biliary cirrhosisand hepatic porphyria, and pericholangitis, sclerosing cholangitis,hepatic fibrosis and chronic active hepatitis, which have been reportedto occur with a high frequency as complications of inflammatory boweldiseases such as ulcerative colitis and Crohn's disease.

Preferably, test agents will assayed for their ability to reduce orprevent of progress of hepatic necrocytosis and/or accelerate hepaticregeneration. For example, expression levels of Rasp-1, a gene that isupregulated during regeneration of liver tissue, can be monitoredfollowing administration of a test agent. Rasp-1 is described in U.S.Pat. No. 6,027,935, the contents of which are incorporated herein byreference for their description of Rasp-1 sequences, antibodies andassays.

In a preferred embodiment, test agents are screened for efficacy in thetreatment of hepatitis viral infections, particularly infections ofhepatitis B and hepatitis C. Other hepatitis viruses that aresignificant as agents of human disease include hepatitis A, hepatitisdelta, hepatitis E, hepatitis F, and hepatitis G (Coates, J. A. V., et.al., Exp. Opin. Ther. Patents (1995) 5 (8): 747-756). The test agent cancomprise, for example, nucleoside analog antivirals, immunomodulators,immunostimulators (e.g., interferons and other cytokines) or otherimmune system-affecting drug candidates, including, but not limited to,thymic peptides, isoprinosine, steroids, Schiff base-formingsalicylaldehyde derivatives such as Tucaresol, levamisol, and the like(Gish, R. G., et al., Exp. Opin. Invest. Drugs (1995) 4 (2):95-115;Coates, J. A. V., et al., Exp. Opin. Ther. Patents (1995) 5(8):747-765).

Anti-hepatitis efficacy of a test agent can be determined according tomethods known in the art. For example, following treatment with a testagent, the amount of hepatitis virus or viral DNA in the culture mediumcan be determined by PCR analysis (e.g., of sedimented particles). DNAmeasurements can be correlated with viral replication to assesspost-treatment infectivity. Alternatively, viral loads can be measureddirectly. Other measures of efficacy include measurement of enzymelevels, including but not limited to SGOT, ALT and LDH, histologicanalysis and normal production of total liver proteins, such as theclotting factors.

In a preferred embodiment, the efficacy of a test agent is determined inliver tissues infected with the hepatitis C virus.

In a preferred embodiment, test agents are screened for efficacy in thetreatment of liver cancer. Reduction or elimination of transformed livercells in response to treatment with a test agent can be detected bymeasuring decreases in hypercalcaemia and CEA expression. Reduction inproliferation can also be determined by cell counting.

C. Combination Three-Dimensional Systems

Three-dimensional systems of the invention can be connected in series toevaluate drug toxicity and efficacy in multiple systems. Preferably, thecombination three-dimensional system would comprise a liver unit. Evenmore preferred is a combination system that comprises an interconnectedliver unit and kidney unit. Thus, the effect of a test agentadministered to the liver unit can additionally be assayed for itsdirect and/or indirect effect on the kidney unit. Most preferred is acombination system that comprises an interconnected liver unit, kidneyunit and cardiac unit.

2. Kidney

A. Toxicity

Toxicity in the kidney can occur, for example, as a result of allergicor hypersensitive immune responses to a test agent. The appearance ofexcess protein, such as albumin and creatinine, in the urine isindicative of toxicity. Thus, in kidney tissue engineered systems of theinvention, assays to detect toxicity of a particular test agentpreferably comprise measurement of proteins including, but not limitedto albumin and creatinine.

B. Efficacy

The kidney is a complex organ with an intricate vascular supply and atleast 15 different cell types, which performs the critical functions offiltration, reabsorption and excretion. The basic functional unit of thekidney, the nephron, is composed of a vascular filter, the glomerulus,and a resorptive unit, the tubule. Filtration is dependent on flow andspecialized glomerular endothelial cells. The majority (50-65%) ofreabsorption is performed by the proximal tubule cells using activesodium transport through the energy-dependent Na⁺—K⁺-ATPase located onthe basolateral membrane. Only 5-10% of the approximately one millionnephrons in each human kidney is required to sustain normal excretoryfunction.

Test agents can be screened for efficacy in tissue engineered systems ofthe invention comprising kidney cells affected with diseases including,but not limited to, glomerulonephritis, ischemia reperfusion injury;bacterial and viral glomerulonephritides, IgA nephropathy andHenoch-Schonlein Purpura, membranoproliferative glomerulonephritis,membranous nephropathy, Sjogren's syndrome, diabetic nephropathy,nephrotic syndrome (minimal change disease, focal glomerulosclerosis andrelated disorders), acute renal failure, acute tubulointerstitialnephritis, pyelonephritis, genetic renal disease (medullary cystic,medullar sponge, polycystic kidney disease (autosomal dominantpolycystic kidney disease, autosomal recessive polycystic kidneydisease, tuborous sclerosis), von Hippel-Lindau disease, familialthin-glomerular basement membrane disease, collagen III glomerulopathy,fibronectin glomerulopathy, Alport's syndrome, Fabry's disease,Nail-Patella Syndrome, congenital urologic anomalies), monoclonalgammopathies (multiple myeloma, amyloidosis and related disorders),febrile illness (familial Mediterannean fever, HIV infection),inflammatory disease (systemic vasculitides, polyarteritis nodosa,Wegener's granulomatosis, polyarteritis, necrotizing and crescenticglomerulonephritis), bacterial infection, allergies and congenitaldefects.

The kidney is able to repair damage to the proximal tubule epitheliumthrough a complex series of events involving cell death, proliferationof surviving proximal tubule epithelial cells, formation of poorlydifferentiated regenerative epithelium over the denuded basementmembrane, and differentiation of the regenerative epithelium to formfully functional proximal tubule epithelial cells (Wallin et al., Lab.Invest. 66:474-484, 1992; Witzgall et al., Mol. Cell. Biol.13:1933-1942, 1994; Ichimura et al., Am. J. Physiol. 269: F653-662,1995; Thadhani et al., N. Engl. J. Med. 334:1448-1460, 1996). KIM genesare upregulated in renal tissue after injury to the kidney, duringkidney regeneration. KIM genes are described in U.S. Pat. No. 6,664,385,the contents of which are incorporated herein by reference for theirdescription of DNA sequences, antibodies and assays. Preferably, testagents will assayed for their ability to reduce or prevent of progressof renal failure and/or accelerate renal regeneration. For example,expression levels genes that are upregulated during regeneration ofkidney tissue, such as the KIM genes, can be monitored followingadministration of a test agent. As another example, total proteinlevels, albumin levels, restored sodium, and clearance of creatinine canbe monitored following administration of a test agent. Clearance oftracer molecules, such as inulin, diethylene-triaminepentaacetic acidand ^(99m)Tc can also be monitored as indicator of the clearance ofother molecules following administration of a test agent.

3. Heart

A. Toxicity

The toxic effect of a test agent in cardiac tissue engineered systems ofthe invention can be detected using a variety of assays known in theart. For example, assays to detect toxicity of a particular test agentpreferably comprise measurement of QT intervals, changes inelectrophysiology (e.g., changes in K⁺/Ca²⁺ channels) and/or arrhythmiaby T-wave alternans (TWA).

Alternans of the electrocardiogram is defined as a change in amplitudeand/or morphology of a component of the ECG that occurs on anevery-other-beat basis (Walker, M. L. and Rosenbaum, D. S., (2003)Cardiovasc. Res. 57: 599-614). TWA is the beat-to-beat alternation ofT-wave amplitude, and is closely linked to electrical instability in theheart. Beat-to-beat microvolt fluctuation of the T wave can be detectedusing high-resolution electrodes and signal processing techniques (Gold,M. R., and Spencer, W. (2003) Curr. Opin. Cardiol. 18: 1-5). A largenumber of beats, generally 128, are sampled, and the voltages ofmultiple corresponding points on the T-wave are computed and averaged.Through fast-Fourier transformation, these consecutive amplitudes aredisplayed spectrally, yielding several frequency peaks. These peakscorrespond to thoracic excursions with respiration, other repetitivebody movements, and ambient electrical noise. The peak at 0.5cycles/beat, if present, is caused by TWA. The alternans magnitude,V_(alt), represents the difference between the even or odd beat and themean amplitude, in microvolts. A threshold of 1.9 uV is used forsignificance. The alternans ratio (k) is another parameter measured andrepresents the ratio of the alternans amplitude to the SD of thebackground noise. It is required to be greater than 3.0 forsignificance. Additionally, TWA must be sustained for more than oneminute.

B. Efficacy

Test agents can be screened for efficacy in tissue engineered systems ofthe invention comprising cardiac cells affected with diseases including,but not limited to, congestive heart failure, coronary artery disease,myocardial infarction, myocardial ischemia, effects of atherosclerosisor hypertension, cardiomyopathy, cardiac arrhythmias, musculardystrophy, muscle mass abnormalities, muscle degeneration, myastheniagravis, infective myocarditis, drug- and toxin-induced muscleabnormalities, hypersensitivity myocarditis, autoimmune endocarditis,and congenital heart disease. Preferably, test agents will assayed fortheir ability to accelerate cardiac regeneration. In general, efficacycan be indicated by detection of improved contractility,electromechanical conduction and/or association, susceptibility toelectrical dysfunction, ventricular fibrillation (sudden death),ionotropy, chronotropy, and decreased leakage of enzymes (e.g., CPK andSGOT).

4. Bone Marrow

A. Toxicity

The toxic effect of a test agent in bone marrow engineered systems ofthe invention can be detected primarily by monitoring the effect of theagent on stem cell production. Stem cell production can be monitored bymethods well known in the art, such as FACS analysis. Stem cells to bemonitored include, but are not limited to hematopoietic progenitors,lymphoid progenitors and myeloid progenitors. In addition, the toxiceffect of a test agent in bone marrow engineered systems of theinvention can be detected by screening for the development of adversesecondary effects, such as B12 deficiency, pernicious anemia andmaturation arrest (failure to divide). Bone marrow engineered systems ofthe invention can also be used as an indicator system for thedevelopment of autoimmune responses. Adverse autoimmune responses willresult in the production of antibodies against albumin-drug conjugates.Suspected adverse autoimmune responses in patients could be confirmed byassaying for the undesired albumin-drug conjugates in bone marrowengineered systems of the invention.

B. Efficacy

Preferably, test agents of the invention can be screened for theirability to increase or decrease production of specific stem cellprogenitors, and the differentiated progeny thereof, including, but notlimited to erythrocytes, platelets, neutrophils, T cells, B cells,eosinophils, basophils, neutrophils, and monocytes. Alternatively, testagents of the invention can be screened for their ability to improve thefunction of sub-optimal marrow. For example, improvement in bone marrowproliferation can be monitored by cell counting methods known in theart.

5. Cartilage

A. Toxicity

The toxic effect of a test agent in cartilage tissue engineered systemsof the invention can be detected using a variety of assays known in theart. Toxicity in cartilage involves abnormal growth, altered metabolicfunction (e.g., glucose metabolism), protein production and alteredhistology. For example, test agents known to have adverse effects oncartilage in developing subjects (e.g., children) comprise a family ofanti-bacterial agents known as the fluoroquinolones. Althoughfluoroquinolones are likely to possess extremely useful anti-microbialproperties, they are potentially harmful to cartilage, and must becarefully screened for toxicity. Methods of the invention can be appliedto the screening of test agents, such as test agents comprisingfluoroquinolones, to identify those that do not cause toxicity incartilage.

Three-Dimensional Systems of the Invention

Three-dimensional systems of the invention are described in U.S. Ser.No. 10/187,247, filed Jun. 28, 2002; Ser. No. 09/560,480, filed Apr. 28,2000, now U.S. Pat. No. 6,455,311; U.S. Ser. No. 10/038,891, filed Jan.2, 2002; and PCT/US03/29880; filed on Sep. 23, 2003, now U.S. Ser. No.10/528,737, filed Mar. 22, 2005, the contents of which are incorporatedherein by reference for their detailed descriptions, figures andexamples, which describe the structure and function of three-dimensionaltissue engineered systems. Descriptions of these systems are alsoreiterated below.

Manufacture of Molds and Polymer Scaffolds

For purposes of this invention a “mold” is a device on the surface ofwhich the branching structure of the microchannels is etched or formed.Fabrication of a mold begins by selection of an appropriate substrate.The choice of a substrate material is guided by many considerations,including the requirements placed on the fabrication process by thedesired mold dimensions, the desired size of the ultimate template, andthe surface properties of the wafer and their interaction with thevarious cell types, extracellular matrix (“ECM”) and polymeric backbone.Also important are the thermal properties, such as the glass transitiontemperature (Tg), which must be high enough so that the network of poresin the mold does not collapse upon solvent removal.

Molds of the present invention can comprise a variety of materials,including, but not limited to, inert materials such as silicon, polymerssuch as polyethylene vinyl acetate, polycarbonate, and polypropylene,and materials such as a ceramic or material such as hydroxyapatite. Inparticular, the mold can comprise from metals, ceramics, semiconductors,organics, polymers, and composites. These materials are eitherinherently suitable for the attachment and culture of animal cells orcan be made suitable by coating with materials described herein toenhance cell attachment and culture (e.g. gelatin, matrigel, vitrogenand other tissue culture coatings known in the art).

In an alternative embodiment, MEMS replica molding can be used to make a“polymer scaffold” for seeding cells. In this method, a mold is made asdescribed herein, preferably of silicon, and is then used as a templateon which a polymeric material is cast. Optionally, the polymer scaffoldcan then be peeled away from the mold and seeded with cells.

A “tissue-defining surface” is the surface of a mold or a polymerscaffold, and a “substrate” is the mold or polymer scaffold itself.

The term “polymer” includes polymers and monomers that can bepolymerized or adhered to form an integral unit. The polymer can benon-biodegradable or biodegradable, typically via hydrolysis orenzymatic cleavage. For implantation, polymer scaffolds are preferablyused, which can be biodegradable polymer scaffolds. For embodimentsrelating to extracorporeal support devices, biocompatible, nondegradablepolymers may facilitate size reduction.

In one embodiment, the biodegradable polymer scaffold comprisesbiodegradable elastomers formed from hydrolyzable monomers as describedin Wang et al, Nature Biotech 20, 602 (2002), the contents of which areincorporated herein by reference. These biodegradable elastomers areanalogous to vulcanized rubber in that crosslinks in a three-dimensionalnetwork of random coils are formed. These biodegradable elsatomers arehydrolyzed over time, preferably within 60 days.

Polymer material for implantation should be selected forbiocompatibility. Any degradation products should also be biocompatible.Relatively high rigidity is advantageous so that the polymer scaffoldcan withstand the contractile forces exerted by cells growing within themold. A biocompatible degradable polymer and its degradation productsare non-toxic toward the recipient.

The term “biodegradable” refers to materials that are bioresorbableand/or degrade and/or break down by mechanical degradation uponinteraction with a physiological environment into components that aremetabolizable or excretable, over a period of time from minutes to threeyears, preferably less than one year, while maintaining the requisitestructural integrity. As used in reference to polymers, the term“degrade” refers to cleavage of the polymer chain, such that themolecular weight stays approximately constant at the oligomer level andparticles of polymer remain following degradation. The term “completelydegrade” refers to cleavage of the polymer at the molecular level suchthat there is essentially complete loss of mass. The term “degrade” asused herein includes “completely degrade” unless otherwise indicated.

Materials suitable for polymer scaffold fabrication include, but are notlimited to, poly-dimethyl-siloxane (PDMS), poly-glycerol-sebacate (PGS),polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid(PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide(PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxidecopolymers, modified cellulose, collagen, polyhydroxybutyrate,polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid),polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyaminoacids, polyorthoesters, polyacetals, polycyanoacrylates, degradableurethanes, aliphatic polyesterspolyacrylates, polymethacrylate, acylsubstituted cellulose acetates, non-degradable polyurethanes,polystyrenes, polyvinyl chloride, polyvinyl fluoride, polyvinylimidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinylalcohol, Teflon®, nylon silicon, and shape memory materials, such aspoly(styrene-block-butadiene), polynorbornene, hydrogels, metallicalloys, and oligo(ε-caprolactone)diol as switchingsegment/oligo(p-dioxyanone)diol as physical crosslink. Other suitablepolymers can be obtained by reference to The Polymer Handbook, 3rdedition (Wiley, N.Y., 1989). Combinations of these polymers may also beused.

Polylactide-co-glycolides (PLGA), as well as polylactides (PLA) andpolyglycolides (PGA) have been used to make biodegradable implants fordrug delivery. See U.S. Pat. No. 6,183,781 and references cited therein.Biodegradable materials have been developed for use as implantableprostheses, as pastes, and as templates around which the body canregenerate various types of tissue. Polymers that are both biocompatibleand resorbable in vivo are known in the art as alternatives to autogenicor allogenic substitutes. In a preferred embodiment, polymers areselected based on the ability of the polymer to elicit the appropriatebiological response from cells, for example, attachment, migration,proliferation and gene expression.

Solvents for most of the thermoplastic polymers are known, for example,methylene chloride or other organic solvents. Organic and aqueoussolvents for protein and polysaccharide polymers are also known. Thebinder can be the same material as is used in conventional powderprocessing methods or can be designed to ultimately yield the samebinder through chemical or physical changes that occur as a result ofheating, photopolymerization, or catalysis.

Properties of the mold and/or polymer scaffold surface can bemanipulated through the inclusion of materials on the mold or in polymerscaffold material which alter cell attachment (for example, by alteringthe surface charge or structure), porosity, flexibility or rigidity(which may be desirable to facilitate removal of tissue constructs).Moreover, advances in polymer chemistry can aid in the mechanical tasksof lifting and folding as well as the biologic tasks of adhesion andgene expression.

For example, molds can be coated with a unique temperature-responsivepolymer, poly-N-isopropyl acrylamide (PNIPAAm), which demonstrates afully expanded chain conformation below 32° C. and a collapsed, compactconformation at high temperatures. When grafted onto surfaces of siliconwafers using electron beam irradiation, it can be used as a temperatureswitch for creating hydrophilic surfaces below 32° C. and hydrophobicsurfaces above 32° C. Since PNIPAAm is insoluble in water over the lowercritical solution temperature (LCST about 32° C.) and reversiblysolubilized below the LCST, cells detach from the substratum by simplylowering the temperature below the LCST. One of skill in the art can 1)engraft the polymer on silicon wafers that are pre-coated withpolystyrene or 2) engraft the polymer on silicon wafers whose surface isfirst modified by vinyl-tricholorosilane. Either of these techniqueswill ensure that the polymer is better integrated and conjugated to itssubstratum (polystyrene in the former case and vinyl groups in the latercase) so that it can serve as an effective thermal switch, useful inreversing cell attachment and detachment as a single contiguous layer ofcells without the usual cell damage.

Another system for promoting both cellular adhesion and lifting of cellsas intact sheets can involve the use of RGD (Arg-Gly-Asp) peptides. TheRGD sequence is part of the domain within the fibronectin molecule thatendows it with the ability to interact with adhesion molecules presenton the cell surface of fibroblasts. Fibronectin itself is awell-characterized extracellular, structural glycoprotein whichinteracts strongly with other extracellular matrix molecules and whichcauses the attachment and spreading of most cells. This function of thefibronectin molecule is localized primarily to the RGD sequence. One ofskill in the art can synthesize RGD peptides with a structural backboneof PMMA that has an RGD peptide sequence at its tips, bound to oneanother with the intermediate layering of polyethylene oxide. Thisallows differential cell adhesion in only selected areas and not others.Once the tissue of desired quality is formed, release of this intactmonolayer of tissue from its substratum is straightforward; it requiresonly the addition of soluble RGD to the culture medium to act as acompetitive substrate to the insolubilized RGD substrate on the siliconmold surface.

Attachment of the cells to the mold and/or polymer scaffold can beenhanced by coating the substrate with compounds such as basementmembrane components, agar, agarose, gelatin, gum arabic, types I, II,III, IV, and V collagen, fibronectin, laminin, glycosaminoglycans,matrigel, vitrogen, mixtures thereof, and other materials known to thoseskilled in the art of cell culture.

Thus, by the methods of the invention, cells can be grown on molds thatare uncoated or coated as described herein, depending upon the materialused for mold construction. Alternatively, cells can be grown on polymerscaffolds made by replica molding techniques.

Design of Apparatus

In a preferred embodiment, mold and/or polymer scaffold pieces arefitted together and optionally separated by a semi-permeable membrane.The vascular cells can be seeded into one layer and cultured to formvascular channels based on the pattern etched in the surface of themold. Organ or tissue specific cells can be added to the secondpatterned surface, where they attach and proliferate to form avascularized tissue bilayer. The second patterned surface optionallycomprises inlets for neural innervation, urine flow, biliary excretionor other activity.

Channel designs can be incorporated into a matrix, with each designcorresponding to a polymer layer arranged in a repeating pattern inthree dimensions. Fabrication allows for multiple silicon master molds,each master mold with a different channel design with interveningthrough-hole layers, rather than having only inlet and outlet layers atopposing diagonal corners, and has a much more complex and dense arrayof through-holes connecting large numbers of smaller vessels.

FIGS. 1-27 depict the molds, microfabrication of the molds,physiological growth of cells in culture that have been seeded on themicrofabricated molds, and configuration of the molds and/or polymerscaffolds into three-dimensional systems.

Construction of Tissue or Organ Equivalents

Engineered tissue lamina can be systematically folded and compacted intoa three-dimensional vascularized structure. The two-dimensional surfaceof the mold can be varied to aid in the folding and compacting process.For example, the surface can be changed from planar to foldedaccordion-like. It can be stacked into multiple converging plates. Itcould be curvilinear or have multiple projections.

Different types of tissue, or multiple layers of the same type oftissue, can be superposed prior to folding and compacting, to createmore complex or larger structures. For example, a tubular system can belayered onto a vascular system to fabricate glomerular tissue andcollecting tubules for kidneys. Bile duct tubes can be overlaid onvascularized liver or hepatocyte tissue, to generate a bile ductdrainage system. Alveolar or airway tissue can be placed on lungcapillaries to make new lung tissue. Nerves or lymphatics can be addedusing variations of these same general techniques.

Three-dimensional tissue and organ formation can be achieved by theaddition of the second mold or polymer scaffold which allows thefunctional unit of the organ to be added, and likewise allows precisionfor patterning of exocrine outflow. For example, in the liver, theparenchymal cells are hepatocytes and the exocrine system is the biliarysystem. By the addition of the second compartment containing hepatocyesand biliary cells, the functional tissue of the liver can be achievedand biliary excretion can be designed and enfolded.

This patterning can be made more complex with the addition of furtherlayers separated by permeable membranes. Several molds and/or polymerscaffolds, with or without semi-permeable membranes between them, can bestacked in rational arrays to produce complex tissue in 3-dimensionalspace. These layers of molds and/or polymer scaffolds, and optionally,semi-permeable membranes, can be appropriately interdigitated andconnected (e.g. via through-holes) to produce vascular connectionsthrough the depths of the stack, as well as excretory outflow systemsthrough the depths of the tracts.

Stacking Molds and/or Polymer Scaffolds to Achieve Three-Dimensionality.

Extension of the two-dimensional technology into the third dimension canbe accomplished by stacking the two-dimensional layers on top of eachother. This stacking method begins with many molds and/or polymerscaffolds produced by the techniques described in previous sections.Once these molds and/or polymer scaffolds (nominally of the same size)are created, they are lain down or bonded to other separate molds and/orpolymer scaffolds, atop one another. The layers are connected at pointswithin small and/or midsized vessels by vertical links, which serve asthrough-holes extending through the z-axis of the molds and/or polymerscaffolds. The pattern of microchannels on the surface of each mold orpolymer scaffold can differ or be similar to the previous layer,depending upon fluid mechanical considerations. Alignment provided byvertical links generates vessel structures that extend up into the third(vertical) dimension.

By extending this technology as needed, one can move from the presentlyachievable formation of small (˜100 cm²) tissue sheets, each containingone plane of blood vessels, to the formation of perhaps 100 cm³ ofmaterial, enough to build an organ. The process is low-cost, scalable,can be customized for the physiology of a particular patient, and isbased upon currently available microfabrication technology.

Fastening the Stacked Layers.

An aspect of this invention is the fastening or sealing of the polymericmold layers. Preferably, the layers are irreversibly bound beforeimplantation into the host. Depending on the composition of the layeredmaterial, the layers can be sealed by solvent bonding; reflow by heating(40° C.); treating surface with oxygen plasma; or by polymer flow at thesurface. Biocompatible polymer materials maybe bonded together by plasmaactivation to form sealed structures (Jo et al., SPIE 3877, 222 (1999)).The basic process results in bonded layers with channel architectureclosely resembling that obtained with silicon etched molds.

Silicon-Glass Microfluidic Chambers to Test Sealing of Stacks.

Microfluidic tests have been performed that demonstrate that bondedapparatuses are leakproof and support fluid pressures necessary fordynamic cell seeding. One of the most common methods used to sealmicromachined wafers together is anodic bonding, a technique based onthe high concentration of mobile ions in many glasses (Camporese, etal., IEEE Electron. Device Lett. EDL 2, 61 (1981)). This processproduces a permanent seal; fracture testing of silicon-glass anodicallybonded interfaces produces a failure within the bulk of the glass.

Etched wafers maybe bonded together, producing closed lumens suitablefor fluidic experiments. A fluidic test was performed with a mixed-phaseflow of alcohol with 10 μm fluorescent microspheres. An unetchedglass-capping layer was mechanically drilled for inlet and outlet fluidports, and then anodic ally bonded to a silicon wafer plasma-etched withthe TEP-1-geometry. A permanent seal with no leaks was produced,enabling one to obtain highly accurate pressure and flow data.

Alternatively, the multilayer device of the invention can be configuredsuch that each of the layers has an alignment indentation on one surfaceof the layer and an alignment protrusion on the opposing surface ofanother layer. The alignment indentations shaped to mate with thealignment protrusion, so that the layers are held together.

Alternative Methods of Stacking.

To build up the mold and/or polymer scaffold layers by mechanicalassembly, the layers can be mechanically mated using biodegradable ornon-biodegradable barbs, pins, screws, clamps, staples, wires, string,or sutures (See U.S. Pat. No. 6,143,293). With this mechanical assemblyapproach, each prefabricated section can comprise different mold and/orpolymer scaffold material and/or different mold microstructures.Different sections of these can be seeded with cells before assembly.Cells thus be can be embedded into the mold or polymer scaffold byassembling sections around these components. In addition, surfacefeatures on each mold, which are readily fabricated, become part of theinternal microstructure (e.g., molded surface channels become conduitsfor cell infusion, or for blood flow to stimulate angiogenesis). Asurface feature on an individual mold or polymer scaffold will become aninternal feature when another segment is assembled over it. For example,surface features such as channels can be micromachined into a first moldor polymer scaffold layer. When a second mold or polymer scaffold layeris placed atop that a first layer, the micromachined surface featurebecomes an internal feature of the apparatus.

Rolling or Folding to Achieve Three-Dimensionality

An alternate method for achieving three-dimensionality is to generate along strip of polymer mold material, which contains repeating units ofthe blood vessel network along with through-holes, and to fold the moldfilm in a z-fold fashion while aligning the through-holes to oneanother.

The rolling or folding process begins with the generation of a lengthystrip of polymer mold material, which contains a serial array of unitcells each of which is comprised of an array of channels mimicking thevascular network, produced from a wafer mold by molding, embossing, orthe like. These unit cells can be identical or can be different. Theunits are linked to through-holes that provide the vertical channelconnections between horizontal blood vessel layers. Once the polymericscaffold strip has been formed, it is folded in a z-fold fashion, andbonded together so that each fold is attached to the film portions aboveand below it with alignment to the through-holes.

This roll can be of a length to provide sufficient scaffolding materialfor an entire human organ, which can be hundreds or even more multiplesof the area of a single wafer. Each section of the roll is a sheet ofpolymeric mold with closed lumens, or vessels. The vessels in eachfolded section of sheet are connected to a through-hole at the edge ofthe sheet (for example, one on each side, for inlet and outlet bloodflow). During folding, the sheet sections are folded such that thethrough-hole openings align, forming a vessel in the third (z)dimension. The roll can be in the shape of a spiral, helix, jelly rollor other cylindrically shaped objects.

The described three-dimensional tissue structures can then be implantedinto animals or patients by directly connecting the blood vessels toflow into and out of the apparatus. Immediate perfusion of oxygenatedblood occurs, which allows survival and function of the entire livingmass.

In a one embodiment, tissue-engineered liver is formed. Preferably,tissue engineered liver comprises both functioning hepatocytes and bileducts. The biliary system of native liver begins with a minute hexagonalbile canaliculus, which is formed from specialization of the adjacentsurfaces of individual hepatocytes, which are sealed with tightjunctions. These canaliculi are confluent with terminal biliaryductules, which are initially made of squamous cells, but give way tolow cuboidal biliary epithelium as they approach the interlobular bileducts. One liter of bile per day is secreted by hepatocytes and movedout of the liver through this system. There have been previous reportsof the formation of duct-like structures in a variety of long-term invitro and in vivo hepatocyte cultures (Block, et al., J Cell Biol, 132,1133 (1996); Landry, et al., J Cell Biol, 101, 914 (1985); Mitaka, etal., Hepatology 29, 111 (1999); Nishikawa, et al., Exp Cell Res, 223,357 (1996); Uyama, et al., Transplantation 55, 932 (1993)).

In a yet another embodiment, tissue-engineered kidney is formed.Preferably, tissue engineered kidney comprises functioning proximaltubules. Tissue-engineered kidney functions as a native kidney;glomerular ultrafiltrate can flow from the glomerular endothelium andpasses through a semipermeable membrane into a proximal tubule networkwhere reabsorption occurs.

System for Modeling and Designing Physiological Networks

Three-dimensional systems of the invention can comprise a physiologicalfluidic network having stacked, two-dimensional layers comprised ofblood vessels, wherein small and/or midsized vessels in one layer arevertically connected to small and/or midsized vessels in at least oneadditional layer by vertical links. Methods of integrating the twodimensional networks, in which small and midsized vessels are arrangedto link the networks vertically in a more complex manner, enable highcell densities and a large number of small vessels to be incorporatedinto the three-dimensional structure of the tissue engineeredconstructs.

These three-dimensional designs comprise stacked, folded or rolledseries of two-dimensional layers, with the two-dimensional layersarranged such that large numbers of interconnection points existsbetween layers. Each two-dimensional layer is generated by using acomputational fluid dynamic (CFD) model, which produces a model networkto simulate the critical structure and function of the tissue or organof interest. The CFD model generates multiple, preferably at least two,distinct two-dimensional layers, which are arranged to allow for a verylarge number of vertical interconnects between layers. Within eachtwo-dimensional layer, unit cells are arranged in a hexagonal pattern,and the thickness of each line in the pattern corresponds to the widthof the fluidic channel.

The resulting three-dimensional structure is comprised of a large numberof two-dimensional layers, arranged in a repeating fashion, and arestacked vertically in a total stack of at least 15 layers. This designcan comprise between about 50 and 2000 layers, more preferably betweenabout 100 and 1000 layers and most preferably about 500 layers.Advantageously, such designs have increased space in the lateraldimension, enabling a much larger number of small channels. It enablesat least one order of magnitude but not more than two orders ofmagnitude increase in the number of small channels.

Preferably, the tissue engineered constructs have a small vessel orcapillary capacity in an amount greater than about 2000 capillaries/cc,greater than about 5000 capillaries/cc, greater than about 10,000capillaries/cc, greater than about 15,000 capillaries/cc, up to about100,000 capillaries/cc. Most preferably, the tissue engineeredconstructs having a small vessel or capillary capacity in an amountequal to or greater than about 10,000 capillaries/cc. Tissue engineeredconstructs of the present invention can maintain physiological pressureand fluid velocities in the network, maximum oxygen diffusion length,and vessel size distribution, which for small vessels is between about100-200 microns. Blood vessels of all sizes are oriented along all threeaxes and along all angles in between. Simultaneous matching of allphysiological parameters results from organization of tissues in a truethree-dimensional coordinated fashion.

Three-dimensional systems of the invention can comprise a physiologicalfluidic network having stacked, two-dimensional layers comprised ofblood vessels, wherein small and/or midsized vessels in one layer arevertically connected to small and/or midsized vessels in at least oneadditional layer by vertical links (FIG. 28). In a preferred embodiment,the tissue engineered construct has a small blood vessel capacity of atleast about 10,000 capillaries/cc and the distance between small bloodvessels is less than about 200 microns.

As used herein, the term “vessel” and blood vessel” are interchangeable.

A “small vessel” or “capillary” refers to a blood vessel that is lessthan 20 microns in diameter.

A “midsized vessel” refers to a blood vessel that is between 20 and 100microns in diameter.

“Physiological” refers to the condition of a blood vessel within anormal living system. In context, “physiological” can also refer to thecondition of a tissue or organ within a normal living system. A“condition” refers to one or more parameters, such as pressure, velocityand capacity of blood flow, shear wall stress, hematocrit distributionand distance between vessels, which for small vessels is between about100-200 microns. Data from two physiological systems are described inKassab et al, Am. J. Physiol 265 (1): H350 (1993) and Kassab, Ann BiomedEng 28 (8): 903 (2000), the contents of which are incorporated herein byreference.

A “vertical link” refers to a partial or complete through hole withinone layer that vertically connects at least one second layer. Verticallinks are perpendicular to the layers which they connect. An “inlet” or“outlet” refers to the placement of tubing within a through hole.

A system for modeling and designing physiological networks can beembodied, in whole or in part, in a software program to be executed by ageneral purpose computing device and/or a specific purpose device havingembedded instructions for performing tasks included in said system. Forillustrative purposes, the invention will be described as embodied insoftware programs executed using a general purpose computing device.

FIG. 29 is a diagram illustrating a system configuration 100. As shownin FIG. 29, system 100 may comprise a computing device 105, which may bea general purpose computer (such as a PC), workstation, mainframecomputer system, and so forth. Computing device 105 may include aprocessor device (or central processing unit “CPU”) 110, a memory device115, a storage device 120, a user interface 125, a system bus 130, and acommunication interface 135. CPU 110 may be any type of processingdevice for carrying out instructions, processing data, and so forth.Memory device 115 may be any type of memory device including any one ormore of random access memory (“RAM”), read-only memory (“ROM”), Flashmemory, Electrically Erasable Programmable Read Only Memory (“EEPROM”),and so forth. Storage device 120 may be any data storage device forreading/writing from/to any removable and/or integrated optical,magnetic, and/or optical-magneto storage medium, and the like (e.g., ahard disk, a compact disc-read-only memory “CD-ROM”, CD-ReWritable“CD-RW”, Digital Versatile Disc-ROM “DVD-ROM”, DVD-RW, and so forth).Storage device 120 may also include a controller/interface (not shown)for connecting to system bus 130. Thus, memory device 115 and storagedevice 120 are suitable for storing data as well as instructions forprogrammed processes for execution on CPU 110. User interface 125 mayinclude a touch screen, control panel, keyboard, keypad, display or anyother type of interface, which may be connected to system bus 130through a corresponding input/output device interface/adapter (notshown). Communication interface 135 may be adapted to communicate withany type of external device, system or network (not shown), such as oneor more computing devices on a local area network (“LAN”), wide areanetwork (“WAN”), the internet, and so forth. Interface 135 may beconnected directly to system bus 130, or may be connected through asuitable interface (not shown).

While the above exemplary system 100 is illustrative of the basiccomponents of a suitable system, many variations of the hardwareconfiguration are possible. As described above, system 100 provides forexecuting processes, by itself and/or in cooperation with one or moreadditional devices, that may include programs for modeling and designingphysiological networks according to flow parameters in accordance withthe present invention. System 100 may be programmed or instructed toperform these processes according to any communication protocol,programming language on any platform. Thus, the processes may beembodied in data as well as instructions stored in memory device 115and/or storage device 120 or received at interface 135 and/or userinterface 125 for execution on CPU 110. Exemplary processes will now bedescribed in detail.

Overview of Design Method

Software tools executed on system 100 may be used to design fluidicnetworks appropriate for use as vasculatures in tissue engineeredorgans. A fluidic network may be considered appropriate for use intissue-engineered organs if the network mimics vital behavior of naturalvasculatures. Two kinds of measurable data on blood vessel networks innature may be used for evaluating a fluidic network: measurements of thegeometry of blood vessels, and measurements of the blood flow behaviorin the vessels. Measurements of the geometry of the blood vessels mayinclude vessel diameters, vessel lengths, and the branching pattern ofthe network. Measurements of the blood flow behavior may include flowvelocities, fluidic pressures, and forces exerted by the fluid shearingagainst the vessel wall.

The flow behavior in a single blood vessel may be modeled by a singleequation. The equation used may vary depending on the type of vessel andtype of fluid in question. A network of blood vessels may be modeled bya system of equations, where each vessel is represented by a singleequation. There may be overlap in the equations: for example, if twovessels are connected, the amount of fluid flowing through one (the flowrate) will equal the amount of fluid flowing into the other. Theequations are thus interrelated, and this type of system is known as asystem of “simultaneous” equations. More equations may be added to thesystem to represent constraints on the network, such as requiring thattwo vessels have the same flow rate. Thus, a fluidic network havingthousands of individual vessels may be described by a system ofthousands of simultaneous equations.

A software program for use with system 100 may include steps as follows:receiving a branching pattern, indicating how many vessels there are,how they are connected to each other, and how large the tissue or organsupported by the fluidic network is. The system may set up an equationfor each vessel, keeping track of how the equations are related to eachother. The system may need more information to solve all of theequations. If the geometry is provided to the system, indicating thelength and diameter of each vessel, the software can solve the systemand determine the flow behavior throughout the network. Furthermore,with flow velocities and pressure, the software can solve the system andprovide an optimal diameter for each vessel.

A combination of geometry and flow behavior may be used: for a branchingpattern, flow may be distributed evenly throughout the network; andlimits on the pressures and flow rates may be placed on the network.FIG. 30 illustrates a network designed using this method.

The technique described above accounts for each vessel by a singleequation and the entire system of equations can be solved at once. Thesingle equation for each vessel assumes that the fluid in the vessel isbehaving as a simple fluid. Blood, however, may be modeled as somethingother than a simple fluid: cells and other materials that affect flowproperties may also be taken into account.

Therefore, a program may be provided to accurately model the flow ofblood in networks and to account for these factors. Including theseeffects expands the system of equations from thousands of equations tohundreds of thousands of equations, and advanced techniques may berequired to solve a system of this size.

Physiological Network Topology

In a blood vessel network, vessels larger than ˜20 μm in diameter mayshow a treelike topology. That is, a large vessel may divide into two ormore smaller vessels. Blood vessels smaller than ˜20 μm, including thecapillaries, may be arranged in bundles, where a number of vessels ofthe same size are interconnected. The topology of a vasculature networkcan be described using a numbering system. In accordance with anembodiment of the invention, a diameter-defined Strahler ordering systemcan be used (Jiang et al., Journal Of Applied Physiology 76 (2):882(1994), the contents of which are incorporated herein by reference). Inthis system, vessels may be given order numbers, where the smallestvessels are order 0 and the larger vessels have higher numbers. Usefulinformation can be extracted from the ordered network, such as whichorder vessels are likely to be connected to each other and how manyvessels of each order exist in the network.

Physiological Flow Behavior

There are several properties the flow through a vasculature may need tosatisfy in order to support a living organ. Basic requirements of avascular network may include:

-   -   1. allowing cells to be seeded on the inside of the vessels;    -   2. providing nutrient transport to and from all parts of the        tissue being supported; and    -   3. being able to be implanted without disturbing blood flow to        other organs.

From each basic requirement more specific required characteristics ofthe network can be derived:

-   -   1. The process of seeding cells on the inside of the vessels and        the behavior of those cells may be highly dependent on the shear        stresses applied by the fluid at the wall. A network may be        generated such that the shear throughout the network is constant        to give uniform seeding and growth. This is consistent with        hypothetical and experimental investigations of physiological        vasculature.    -   2. The network may include a sufficient number of vessels        distributed throughout the volume of the tissue to provide        nutrient transport throughout the organ. The distribution of        various-sized vessels has been measured in numerous        physiological systems, and a network may be generated to match        the physiological distribution.    -   3. The network may be generated such that conditions at the        boundary match those of the network being replaced, thus        allowing implantation without disturbing blood flow to other        organs. The boundary conditions are the pressure drop across the        network and the total flow rate through the network. These        values have been measured for various organs in numerous        animals.

A vasculature design satisfying the above-listed criteria may be used tosupport a living organ.

Device Topology Design

The first step of designing a fluidic network is to specify a networktopology. Networks may be described using a node-vessel form often usedto describe networks of electrical resistors. This format may consist ofa list of nodes and a list of vessels. In the list of nodes, an x, y,and z location is assigned to each node. For n nodes the list may be:

$\begin{matrix}{{Nodes} = {{\begin{matrix}\begin{matrix}\begin{matrix}{1x_{1}y_{1}z_{1}} \\{2x_{2}y_{2}z_{2}}\end{matrix} \\\vdots\end{matrix} \\{n\; x_{n}y_{n}z_{n}}\end{matrix}}.}} & (1)\end{matrix}$

A vessel connects two nodes, so in the list of vessels each vessel maybe defined by the two nodes at its ends. For m vessels:

$\begin{matrix}{{{Vessels} = {\begin{matrix}{1\mspace{14mu} {node}_{{1a}\mspace{11mu}}{node}_{1b}} \\{2\mspace{14mu} {node}_{2a}\mspace{14mu} {node}_{2b}} \\\vdots \\{m\; {node}_{ma}\mspace{11mu} {node}_{mb}}\end{matrix}}},} & (2)\end{matrix}$

where node_(ia) is the node at one end of vessel i and node_(ib) is thenode at the other end. Such a network may be created and manipulated ina programming environment on system 100, such as Matlab® and the like.

FIG. 31 is a diagram showing a network, Testnet-1, created by placingnodes on a grid and using an algorithm to connect selected nodes. Thealgorithm may count through all of the nodes on the grid and createvessels to connect each node to its nearest neighbors. Modifications maybe made to the network by manually changing node locations and deletingsome vessels.

A network may be created using a different method. A graphical userinterface, for example, the Matlab® interface, may be used to create adrawing program. Lines drawn by a user are recorded in node-vesselformat. Each time a new line is drawn the distances between itsendpoints and all other existing endpoints are measured. If the distancebetween any two points is short enough, they can be considered to be thesame point.

FIG. 32 is a diagram illustrating a network, Testnet-1, using thismethod.

This network was created to match the branching pattern of aphysiological capillary bed. The network was then multiplied over alarger area and more connections were drawn in, creating the fulltopology of the design shown in FIG. 32.

The topography of a three-dimensional stacked design may be based on afractal algorithm. This algorithm was chosen because it can be used tocreate patterns of any size with the vessels spaced evenly throughout,and avoids sharp angles between intersecting vessels. The algorithm mayinclude steps as follows:

-   -   1. Define a reference direction in the plane.    -   2. Define a vessel length L.    -   3. Define an origin.    -   4. Define a maximum pattern diameter.    -   5. Create three vessels with one node at the origin, each length        L: one extending parallel to the reference direction, one at        120° from the reference direction, and one 240° from the        reference direction.    -   6. Iterate through all of the newly created nodes. At each node,        redefine the reference direction as the direction of a vessel        connected to that node, redefine the origin as the current node,        and perform step 5.    -   7. Repeat steps 5 and 6 until no new nodes can be added within        the maximum pattern diameter.

A two-dimensional pattern produced by this algorithm is shown in FIG.40. A three-dimensional network may be generated by stacking a set oftwo-dimensional layers. Layers in the middle of the pattern may beidentical, while layers on the outside may be less dense to act asdistribution layers. The software may search through all nodes in thenetwork, connecting each pair of nodes that are found on adjacent layersand are aligned vertically. Inlet and outlet vessels may be added toeither end, completing the initial network. A sample initial network isshown in FIG. 41.

FIG. 34 illustrates a three-dimensional design, referred to here as“Hextak.” As shown in FIG. 34, a series of vessel layers may be laid outon different wafers. The vessels in various layers may be interconnectedby a pattern of vertical vessels. These vertical vessels are added tothe design by placing them at regular distances throughout the patternsand are also shown in FIG. 34. The Hextak design may include fourseparate layers of vertical vessels, as shown in FIG. 34. A method forthis Hextak design will be described in further detail below. Theabove-described methods create a network topology and describe it innode-vessel format. The node-vessel format may then be used to model thefluidic behavior in the network.

Predicting Flow Through a Single Microfabricated Vessel

The flow behavior and geometry of a cylindrical blood vessel can berelated by a single equation,

$\begin{matrix}{{{\mu \frac{Q}{\Delta \; P}} = \frac{\pi \; D^{4}}{128\; L}},} & (3)\end{matrix}$

where Q is the flow rate through the vessel, μ is the viscosity of thefluid flowing through the vessel, ΔP is the pressure drop in the vessel,D is the diameter of the vessel, and L is the length of the vessel. Theterms on the left-hand side of Equation (3) are representative of theflow behavior while the terms on the right-hand side are representativeof the geometry of the vessel. Thus, if the geometry of a vessel isknown, the flow behavior can be calculated and if the flow behavior isknown, the geometry can be calculated.

The vessel can be described as a fluidic resistor, analogous to anelectrical resistor. In this case the relation between flow rate andpressure drop may be given by

$\begin{matrix}{{\frac{\Delta \; P}{Q} = R},} & (4)\end{matrix}$

where R is the fluidic resistance of the vessel, which is dependent onlyon the geometry of the vessel and the viscosity of the fluid. Equations3 and 4 can be rearranged to find the resistance of a cylindrical vessel

$\begin{matrix}{R_{i} = \frac{8\mu \; L}{\pi \; D^{4}}} & (5)\end{matrix}$

Similar equations for various non-cylindrical vessels, including etchedvessels, may also be used. The flow in a lithography-etched vessel maybe accurately predicted by the relation for flow in a rectangular vessel

$\begin{matrix}{{{\Delta \; P} = {\frac{\mu \; {L\left( {x + y} \right)}^{2}}{8({xy})^{3}}\left( {96 - {95\left( \frac{x}{y} \right)} + {56\left( \frac{x}{y} \right)^{2}}} \right)Q}},} & (6)\end{matrix}$

where ΔP is the pressure drop from one end of the vessel to the otherend, μ is the viscosity of the fluid flowing through the vessel, L isthe length of the vessel, x is the width of the vessel, y is the depthof the vessel, and Q is the flow rate through the vessel.

Combining Equations (4) and (6) gives the expression for the fluidicresistance of a vessel:

$\begin{matrix}{R = {\frac{8({xy})^{3}}{\mu \; {L\left( {x + y} \right)}^{2}}\left( {96 - {95\left( \frac{x}{y} \right)} + {56\left( \frac{x}{y} \right)^{2}}} \right)^{- 1}}} & (7)\end{matrix}$

Predicting Flow Through a Network of Vessels

As a single vessel can be modeled by a fluidic resistor, a network ofvessels can be modeled by a network of resistors (i.e. an electriccircuit). Thus, methods for analyzing electrical resistor networks maybe used for analyzing analogous fluidic networks.

The node-vessel topology can be translated into a set of equationsdescribing the flow behavior in the network. The first networkcalculation is to find the flow rates throughout the network. Since itis desirable to have the flow throughout a network be evenlydistributed, vessels may be designated as capillaries in size and a flowrate for each may be assigned accordingly. The flow rates may be storedin a list where each vessel has a corresponding flow rate. The equationfor continuity in fluidic networks, or the analogous Kirchoff CurrentLaw in electrical circuits, tells that the sum of all flow rates at anynode in the network is zero. As long as enough capillaries are defined,this law may be used to find the flow rates throughout the network. Thesoftware goes through the list of nodes calculating flow rates at everynode that it can. For example, if a node has three vessels connected toit and two of those vessels have known flow rates, the flow rate in thethird vessel can be found. Thus, the flow rates of all of the vessels inthe network may be determined.

Once all the flow rates are known, a system of equations describing theflow behavior in the network can be created. The network where thepressures and resistances throughout the network are unknown may bedescribed by a set of simultaneous linear equations. In matrix form,

$\begin{matrix}{{{\begin{bmatrix}\begin{matrix}{\lbrack N\rbrack \mspace{14mu}} & \lbrack Q\rbrack\end{matrix} \\\lbrack K\rbrack\end{bmatrix}\begin{bmatrix}\overset{\_}{P} \\\overset{\_}{R}\end{bmatrix}} = \begin{bmatrix}\overset{\_}{0} \\\overset{\_}{k}\end{bmatrix}},} & (8)\end{matrix}$

where [N] is a matrix that picks values out of the pressure vector to goin the element equations, [Q] is a diagonal vector with entriescorresponding to the flowrate in each vessel, P is the vector of unknownpressures, R is the vector of unknown resistances, [K] is a matrix ofvalues representing the constraint equations, 0 is a vector of zeros,and k is a vector of known values.

The upper section of Equation (8),

$\begin{matrix}{{{{\left. 〚N \right\rbrack\left\lbrack Q〛 \right.}\begin{bmatrix}\overset{\_}{P} \\\overset{\_}{R}\end{bmatrix}} = \left\lbrack \overset{\_}{0} \right\rbrack},} & (9)\end{matrix}$

represents a set of Equation (4)'s, one for each vessel. This set ofequations is set up by counting through the list of vessels,representing the two nodes at the end of each vessel in [N], andrepresenting the flow rate in each vessel in [Q].

The bottom section of Equation (8),

$\begin{matrix}{{{\lbrack K\rbrack \begin{bmatrix}\overset{\_}{P} \\\overset{\_}{R}\end{bmatrix}} = \left\lbrack \overset{\_}{k} \right\rbrack},} & (10)\end{matrix}$

represents the constraint equations. The system cannot be solved unlessthe number of rows in the matrix on the left-hand side of Equation (8)is equal to the number of pressures plus the number of resistances, i.e.there are n equations and n unknowns. Equation (9) supplies a number ofrows equal to the number of resistances, so Equation (10) may be used tosupply a number of rows equal to the number of pressures, also equal tothe number of nodes in the system. The constraint equations can setpressures or resistances to constant values, or set relationshipsbetween resistances or relationships between pressures, or anycombination. The pressure at the inlet and outlet of the network mayalways be set to constant values in accordance with physiologicalrequirements. Other relations may be to set pressure fronts where groupsof nodes have the same pressure, or to set resistance groups, such asforcing all capillaries to have the same resistance. Some sets ofconstraints do not yield a solution, but others are guaranteed to alwayshave a solution as long as reasonable numerical values are placed on theconstraints. One of these sets is to constrain all of the pressures inthe network and solve only for resistance.

Equation (7) is asymmetric and must be solved using Sparse GaussianElimination. The result is a list of the pressures and resistancesthroughout the network. When the resistances are known and an etch depthfor each layer is specified, Equation (7) can be used to find the widthof each vessel. Equation (7) cannot be solved explicitly for the widthgiven the value of R, so a Newton Method may be used to solve Equation(7) for the width. Once the width of each vessel is known, the design iscomplete; the orientation and shape of all vessels in the network isknown.

Schematics of the completed designs for Testnet-0 and Testnet-1 areshown in FIG. 36. FIG. 37 is a diagram of a section of Hextak where eachvessel is plotted as an individual three-dimensional element.

Experimental Verification of Fluidic Model

The model of flow in a single vessel and predictions of flows in smallpatterns have been shown to be accurate by measuring the flow ratesthrough individual vessels and small patterns. An example from this workis presented in FIG. 38, showing the ability to predict the fluidicresistance of vessels with varying widths or varying lengths. The modelwas also shown to be accurate in small patterns in predicting flows atvarying pressures and flow rates through vessels of varying depths andtopology.

The theoretical model has been shown to accurately predict grossbehavior of full networks. As shown in FIG. 39, the model predictionsmatch the experimental behavior within an acceptable error for variationin the elasticity of the material used to construct the network.

Implementation of Design Method

The design method may be implemented in any programming language, suchas the C programming language and so forth. Programming code may becompiled using the GNU compiler collection. As described before, system100 may include any general purpose computing device. For example,compiling and computation may be performed on an SGI Origin 2000workstation. Graphics may be plotted using Matlab, Tecplot, or the like.

The design method may include steps as follows:

-   -   1. Generate a network topology in Node-Vessel format using a        fractal method.    -   2. Generate the system of flow equations (Equation 9).    -   3. Set the system of constraint equations (Equation 10).    -   4. Solve all of the equations (Equation 8) simultaneously to        determine the vessel resistances.    -   5. Use the resistance-geometry relation (Equation 4) to        determine the geometry of each vessel.

A full network appropriate for use as support for a tissue engineeredorgan is shown in FIG. 42.

Comparison of Network Designs to Physiological Systems

Once a network design has been completed, the Strahler ordering systemcan be applied to compare the network to physiological data. For thediameter-defined Strahler system, the diameter distribution of adesigned network may match that of measured physiological systems.

Flow properties may be used as inputs to the design so that the flowproperties throughout the network also match physiological values. Thenumber of vessels of each order may be compared. Mass transport occursin the smallest vessels, so a network must have the appropriate numberof small vessels to viably support an organ. A highly interconnectedstacked device such as the Hextak provides the necessary number ofvessels.

Micromachining and Chemical Processing of Silicon and Other MoldMaterials

Molds can be made by creating small mechanical structures in silicon,metal, polymer, and other materials using microfabrication processes.These microfabrication processes are based on well-established methodsused to make integrated circuits and other microelectronic devices,augmented by additional methods developed by workers in the field ofmicromachining.

Microfabrication processes that can be used in making the moldsdisclosed herein include lithography; etching techniques, such aslasers, plasma etching, photolithography, or chemical etching such aswet chemical, dry, and photoresist removal; or by solid free formtechniques, including three-dimensional printing (3DP),stereolithography (SLA), selective laser sintering (SLS), ballisticparticle manufacturing (BPM) and fusion deposition modeling (FDM); bymicromachining; thermal oxidation of silicon; electroplating andelectroless plating; diffusion processes, such as boron, phosphorus,arsenic, and antimony diffusion; ion implantation; film deposition, suchas evaporation (filament, electron beam, flash, and shadowing and stepcoverage), sputtering, chemical vapor deposition (CVD), epitaxy (vaporphase, liquid phase, and molecular beam), electroplating, screenprinting, lamination or by combinations thereof. See Jaeger,Introduction to Microelectronic Fabrication (Addison-Wesley PublishingCo., Reading Mass. 1988); Runyan, et al., Semiconductor IntegratedCircuit Processing Technology (Addison-Wesley Publishing Co., ReadingMass. 1990); Proceedings of the IEEE Micro Electro Mechanical SystemsConference 1987-1998; Rai-Choudhury, ed., Handbook of Microlithography,Micromachining & Microfabrication (SPIE Optical Engineering Press,Bellingham, Wash. 1997). The selection of the material that is used asthe mold determines how the surface is configured to form the branchingstructure. The following methods are preferred for making molds.

Typically, micromachining is performed on standard bulk single crystalsilicon wafers of a diameter ranging between about 50 and 300millimeters (mm), preferably approximately 100 mm, and of thicknessranging between about 200 and 1200 μm. These wafers can be obtained froma large number of vendors of standard semiconductor material, and aresawn and polished to provide precise dimensions, uniformcrystallographic orientation, and highly polished, optically flatsurfaces. Wafers made from pyrex borosilicate or other glasses can alsobe procured and inserted into micromachining processes, with alternativeprocesses used to etch the glassy materials.

The geometry of the mold, in particular the number of different featuredepths required, is the major factor determining the specific processsequence. The simplest case is that of a single depth dimension for themold. Specifically, for a silicon substrate, the process sequence is asfollows: first, the silicon wafer is cleaned, and a layer ofphotosensitive material is applied to the surface. Typically, the layeris spun on at a high revolution rate to obtain a coating of uniformthickness. The photoresist is baked, and the wafer is then exposed toultraviolet or other short-wavelength light though a semi-transparentmask. This step can be accomplished using any one of several maskingtechniques, depending on the desired image resolution. The resist isthen developed in an appropriate developer chemistry, and the wafer isthen hard-baked to remove excess solvent from the resist. Once thelithographic process has been completed, the wafer can be etched in aplasma reactor using one of several possible chemistries. Etching servesto transfer the two-dimensional pattern into the third dimension: aspecified depth into the wafer. Plasma parameters are determined by thedesired shape of the resulting trench (semi-circular, straight-walledprofile, angled sidewall), as well as by the selectivity of the etchantfor silicon over the masking photoresist. Once the etching has beencompleted, the photoresist can be removed and the wafer prepared for usein the tissue molding process.

Increased flexibility in the geometry of wafer mold can be obtained byinserting additional cycles of masking and etching, as shown in FIG. 1.Here, a second step in which a masking layer has been applied, and openareas etched, is shown. This modification provides the opportunity tomachine channels of varying depths into the wafer mold. To design a moldthat is suitable for the culturing of endothelial cells, increasedflexibility is very important due to the need for vascular branches withdifferent diameters. The techniques can be extended to provide as manyadditional layers and different depths as are desired. In addition,these techniques can be used to create secondary patterns within thepattern of microchannels. For example, it may be advantageous to havewells within the microchannels for culturing additional cell types suchas feeder cells. The pattern of microchannels also can be designed tocontrol cell growth, for example, to selectively control thedifferentiation of cells.

Glass and polymeric wafer molds can be fabricated using a similarsequence, but the actual process can be modified by the addition of anintervening masking layer, since etchants for these materials may attackphotoresist as well. Such intervening materials simply function totransfer the pattern from the photoresist to interlayer and then on tothe wafer below. For silicon etched in one of several wet chemistries,an intervening layer may also be necessary.

The size distribution of the etched porous structure is highly dependenton several variables, including doping kind and illumination conditions,as detailed in Lehmann, “Porous Silicon—A New Material for MEMS”, IEEEProceedings of the Micro Electro Mechanical Systems Conference, pp. 1-6(1996). Porous polymer molds can be formed, for example, by micromoldinga polymer containing a volatilizable or leachable material, such as avolatile salt, dispersed in the polymer, and then volatilizing orleaching the dispersed material, leaving a porous polymer matrix in theshape of the mold. Hollow molds can be fabricated, for example, usingcombinations of dry etching processes (Laermer, et al., “Bosch DeepSilicon Etching: Improving Uniformity and Etch Rate for Advanced MEMSApplications,” Micro Electro Mechanical Systems, Orlando, Fla., USA,(Jan. 17-21, 1999); Despont, et al., “High-Aspect-Ratio, Ultrathick,Negative-Tone Near-UV Photoresist for MEMS”, Proc. of IEEE 10^(th)Annual International Workshop on MEMS, Nagoya, Japan, pp. 518-522 (Jan.26-30, 1997)); micromold creation in lithographically-defined polymersand selective sidewall electroplating; or direct micromolding techniquesusing epoxy mold transfers.

Polymeric molds can also be made using microfabrication. For example,the epoxy molds can be made as described above, and injection moldingtechniques can be applied to form the structures. These micromoldingtechniques are relatively less expensive to replicate than the othermethods described herein.

Three-dimensional printing (3DP) is described by Sachs, et al.,Manufacturing Review 5, 117-126 (1992) and U.S. Pat. No. 5,204,055 toSachs, et al. 3DP is used to create a solid object by ink-jet printing abinder into selected areas of sequentially deposited layers of powder.Each layer is created by spreading a thin layer of powder over thesurface of a powder bed. The powder bed is supported by a piston, whichdescends upon powder spreading and printing of each layer (or,conversely, the ink jets and spreader are raised after printing of eachlayer and the bed remains stationary). Instructions for each layer arederived directly from a computer-aided design (CAD) representation ofthe component. The area to be printed is obtained by computing the areaof intersection between the desired plane and the CAD representation ofthe object. The individual sliced segments or layers are joined to formthe three-dimensional structure. The unbound powder supports temporarilyunconnected portions of the component as the structure is built but isremoved after completion of printing.

SFF methods other than 3DP that can be utilized to some degree asdescribed herein are stereo-lithography (SLA), selective laser sintering(SLS), ballistic particle manufacturing (BPM), and fusion depositionmodeling (FDM). SLA is based on the use of a focused ultra-violet (UV)laser that is vector scanned over the top of a bath of aphotopolymerizable liquid polymer material. The UV laser causes the bathto polymerize where the laser beam strikes the surface of the bath,resulting in the creation of a first solid plastic layer at and justbelow the surface. The solid layer is then lowered into the bath and thelaser generated polymerization process is repeated for the generation ofthe next layer, and so on, until a plurality of superimposed layersforming the desired apparatus is obtained. The most recently createdlayer in each case is always lowered to a position for the creation ofthe next layer slightly below the surface of the liquid bath. A systemfor stereolithography is made and sold by 3D Systems, Inc., of Valencia,Calif., which is readily adaptable for use with biocompatible polymericmaterials. SLS also uses a focused laser beam, but to sinter areas of aloosely compacted plastic powder, the powder being applied layer bylayer. In this method, a thin layer of powder is spread evenly onto aflat surface with a roller mechanism. The powder is then raster-scannedwith a high-power laser beam. The powder material that is struck by thelaser beam is fused, while the other areas of powder remain dissociated.Successive layers of powder are deposited and raster-scanned, one on topof another, until an entire part is complete. Each layer is sintereddeeply enough to bond it to the preceding layer. A suitable systemadaptable for use in making medical devices is available from DTMCorporation of Austin, Tex.

BPM uses an ink-jet printing apparatus wherein an ink-jet stream ofliquid polymer or polymer composite material is used to createthree-dimensional objects under computer control, similar to the way anink jet printer produces two-dimensional graphic printing. The mold isformed by printing successive cross-sections, one layer after another,to a target using a cold welding or rapid solidification technique,which causes bonding between the particles and the successive layers.This approach as applied to metal or metal composites has been proposedby Automated Dynamic Corporation of Troy, N.Y. FDM employs an x-yplotter with a z motion to position an extrudable filament formed of apolymeric material, rendered fluid by heat or the presence of a solvent.A suitable system is available from Stratasys, Incorporated ofMinneapolis, Minn.

The design of the channels in the mold can be constructed by a number ofmeans, such as fractal mathematics, which can be converted by computersinto two-dimensional arrays of branches and then etched onto wafers.Also, computers can model from live or preserved organ or tissuespecimens three-dimensional vascular channels, convert totwo-dimensional patterns and then help in the reconversion to athree-dimensional living vascularized structure. Techniques forproducing the molds include techniques for fabrication of computer chipsand microfabrication technologies. Other technologies include lasertechniques.

Semi-Permeable Membrane

A semi-permeable membrane can be used to separate the first mold orpolymer scaffold from the second mold or polymer scaffold in themicrofabricated apparatuses of the invention. Preferably, the pore sizeof the membrane is smaller than the cell diameters, thus, cells will notbe able to pass through (i.e. a low permeability for animal cells),while low molecular weight nutrients and fluids can pass through (i.e. ahigh permeability for nutrients), thereby providing adequatecell-to-cell signaling. Cell sizes vary but in general, they are in therange of microns. For example, a red blood cell has a diameter of 8 μm.Preferably, the average membrane pore size is on a submicron-scale toensure effective screening of the cells.

In specific embodiments, the semi-permeable membrane will contain anendothelial layer, in order to inhibit passage of metabolites betweenlayers. For example, a microfabricated apparatus could comprise bilayerunits, each layer separated by an endothelized membrane, wherein anupper layer comprises vasculature and a lower layer comprises livercells. Upon application to the vasculature, the test agent wouldcirculate through flow connectors into the lower layer, where it ismetabolized. Metabolites would then flow out of the system with the bile(e.g., through bile ducts).

The endothelial lining of the semi-permeable membrane can comprise extracellular matrix components for support. Alternatively, a polymer layerthat does not inhibit flow between layers, such as a polycarbonate orpolyethersulfone layer, can be applied. Semi-permeable membranes of thepresent invention comprise a wide array of different membrane types andmorphologies, which can be classified as follows:

-   -   (1) Track-etch membranes consisting of cylindrical through-holes        in a dense polymer matrix. These membranes are typically made by        ion-etching; or    -   (2) Fibrous membranes made by various deposition techniques of        polymeric fibers. While these membranes do not have a        well-defined pore topology, production methods have been        sufficiently refined so that fibrous membranes have specific        molecular weight cut-offs.

Track-etch type membranes are preferred, as they limit the fluid motionin one direction. Preferably, fluid motion is in the vertical direction.Fibrous membranes permit fluid motion both laterally and vertically.

The development of an appropriate membrane will mirror the deviceprogression. Biocompatible and non-degradable membranes can beincorporated in microchannels that are made from poly(dimethyl siloxane)(PDMS). Since PDMS is non-degradable, the membranes do not need to bedegradable either. However, degradable membranes and materials formicrochannels can also be used. There exists a variety of commercialtrack-etch membranes with well-defined pore sizes that can be used forthis purpose. Care must be taken to properly incorporate the membranesinto the existing microchannels without leaking. To this end, themembranes can be bonded with either an oxygen plasma or a silicone-basedadhesive. A small recession can be designed into the microchannels sothat the membrane can fit tightly therein.

In principle, membrane formation from polymers relies on phase-phaseseparation. Polymer-solvent interactions are complex, and polymer phasediagrams are significantly more complicated than those for monomericmaterials, e.g., metals. Phase separation can be induced either bydiffusion (diffusion-induced phase separation or “DIPS”) or by thermalmeans (thermal induced phase separation or “TIPS”).

A DIPS system comprises polymer, solvent and non-solvent. The polymersolution is cast as a thin film and then immersed in a coagulation bathcontaining the non-solvent. This process is governed by the diffusion ofvarious low molecular weight components. The exchange of solvent andnon-solvent between the polymer solution and the coagulation bath leadsto a change in the composition in the film and phase separation isinduced. After some time, the composition of the polymer-rich phasereaches the glass transition composition and the system solidifies. Toavoid macrovoid formation, a small amount of non-solvent can be mixedwith the polymer solution. In a preferred embodiment, the polymer ispolycaprolactone (PCL) and the separation system is chloroform/methanol.Specifically, a polymer solution with a concentration ranging from about5-10% wt. is made. PCL is prepared by dissolving it in chloroform atroom temperature under gentle stirring. Once the polymer has completelydissolved, a small amount is placed on a clean mirror surface, and amembrane knife is used to spread out a film with preset thickness. Thethickness of the film can be adjusted by changing the gap between theknife blade and the mirror surface. Once the film has been spread, theentire mirror is immersed in a methanol bath. Phase separation occursalmost instantaneously, but the film and mirror are left in thecoagulation bath for up to about 10 minutes to lock in the morphology. Atypical membrane thickness is about 100 μm, and the pore size is on theorder of about 1 μm, preferably between about 0.01 and 20 μm. Membranemorphology can be varied by altering the composition/concentration ofthe polymer solution, the film thickness, the components of thecoagulation bath, and/or the process conditions. One skilled in the artwould understand how to vary any one of these parameters to achieve thedesired result.

A TIPS system comprises a thermal gradient to induce phase separation.By choosing a polymer-solvent system that is miscible at hightemperatures, but immiscible at low temperatures, e.g., roomtemperature, phase separation can be induced upon cooling down thepolymer solution. In a preferred embodiment, the polymer is PCL and theseparation system is DMF/10% C₃H₈O₃.

Cells to be Seeded onto the Mold or Polymer Scaffold

The tissue will typically include one or more types of functional,mesenchymal or parenchymal cells, such as smooth or skeletal musclecells, myocytes (muscle stem cells), fibroblasts, chondrocytes,adipocytes, fibromyoblasts, ectodermal cells, including ductile and skincells, hepatocytes, kidney cells, pancreatic islet cells, cells presentin the intestine, and other parenchymal cells, osteoblasts and othercells forming bone or cartilage, and hematopoietic cells. In some casesit may also be desirable to include nerve cells. The vasculature willtypically be formed from endothelial cells. “Parenchymal cells” includethe functional elements of an organ, as distinguished from the frameworkor stroma. “Mesenchymal cells” include connective and supportingtissues, smooth muscle, vascular endothelium and blood cells.

Cells can be obtained by biopsy or harvest from a living donor, cellculture, or autopsy, all techniques well known in the art. Cells arepreferably autologous. Cells to be implanted can be dissociated usingstandard techniques such as digestion with a collagenase, trypsin orother protease solution and are then seeded into the mold or polymerscaffold immediately or after being maintained in culture. Cells can benormal or genetically engineered to provide additional or normalfunction. Immunologically inert cells, such as embryonic or fetal cells,stem cells, and cells genetically engineered to avoid the need forimmunosuppression can also be used. Methods and drugs forimmunosuppression are known to those skilled in the art oftransplantation.

Undifferentiated or partially differentiated precursor cells, such asembryonic germ cells (Gearhart, et al., U.S. Pat. No. 6,245,566),embryonic stem cells (Thomson, U.S. Pat. Nos. 5,843,780 and 6,200,802),mesenchymal stem cells (Caplan, et al. U.S. Pat. No. 5,486,359), neuralstem cells (Anderson, et al., U.S. Pat. No. 5,849,553), hematopoieticstem cells (Tsukamoto, U.S. Pat. No. 5,061,620), multipotent adult stemcells (Furcht, et al., WO 01/11011) can be used in this invention. Cellscan be kept in an undifferentiated state by co-culture with a fibroblastfeeder layer (Thomson, U.S. Pat. Nos. 5,843,780 and 6,200,802), or byfeeder-free culture with fibroblast conditioned media (Xu, et al. Nat.Biotechnol., 19, 971 (2001)). Undifferentiated or partiallydifferentiated precursor cells can be induced down a particulardevelopmental pathway by culture in medium containing growth factors orother cell-type specific induction factors or agents known in the art.Some examples of such factors are shown in Table 1.

TABLE 1 Selected Examples of Differentiation Inducing Agents AgentProgenitor Differentiated Cell Vascular Endothelial EmbryonicHematopoietic Cell¹ Growth Factor Stem Cell Sonic Hedgehog Floor PlateMotor Neuron² Insulin-like Growth Embryonic Myoblast³ Factor II StemCell Osteogenin Osteoprogenitor Osteoblast⁴ Cytotoxic T Cell Spleen CellCytotoxic T Differentiation Factor Lymyphocyte⁵ β-catenin Skin Stem CellFollicular Keratinocyte⁶ Bone Morphogenic Mesenchymal Adipocytes,Osteoblasts⁷ Protein 2 Stem Cell Interleukin 2 Bone Marrow NaturalKiller Cells⁸ Precursor Transforming Growth Cardiac Cardiac Myocyte⁹Factor β Fibroblast Nerve Growth Factor Chromaffin Cell SympatheticNeuron¹⁰ Steel Factor Neural Crest Melanocyte¹¹ Interleukin 1Mesencephalic Dopaminergic Neuron¹² Progenitor Fibroblast Growth GHFTLactotrope¹³ Factor 2 Retinoic Acid Promyelocytic Granulocyte¹⁴ LeukemiaWnt3 Embryonic Stem Cell Hematopoietic Cell¹⁵ ¹Keller, et al. (1999)Exp. Hematol. 27: 777-787. ²Marti, et al. (1995) Nature. 375: 322-325.³Prelle, et al. (2000) Biochem. Biophy. Res. Commun. 277: 631-638.⁴Amedee, et al. (1994) Differentiation. 58: 157-164. ⁵Hardt, et al.(1985) Eur. J. Immunol. 15: 472-478. ⁶Huelsken, et al. (2001) Cell. 105:533-545. ⁷Ji, et al. (2000) J. Bone Miner. Metab. 18: 132-139.⁸Migliorati, et al. (1987) J. Immunol. 138: 3618-3625. ⁹Eghbali, et al.(1991) Proc. Natl. Acad. Sci. USA. 88: 795-799. ¹⁰Niijima, et al. (1995)J. Neurosci. 15: 1180-1194. ¹¹Guo, et al. (1997) Dev. Biol. 184: 61-69.¹²Ling, et al. (1998) Exp. Neurol. 149: 411-423. ¹³Lopez-Fernandez, etal. (2000) J. Biol. Chem. 275: 21653-60. ¹⁴Wang, et al, (1989) Leuk Res.13: 1091-1097. ¹⁵Lako, et al. (2001) Mech. Dev. 103: 49-59.

A stem cell can be any known in the art, including, but not limited to,embryonic stem cells, adult stem cells, neural stem cells, muscle stemcells, hematopoietic stem cells, mesenchymal stem cells, peripheralblood stem cells and cardiac stem cells. Preferably, the stem cell ishuman. A “stem cell” is a pluripotent, multipotent or totipotent cellthat can undergo self-renewing cell division to give rise tophenotypically and genotypically identical daughter cells for anindefinite time and can ultimately differentiate into at least one finalcell type.

The quintessential stem cell is the embryonal stem cell (ES), as it hasunlimited self-renewal and multipotent and/or pluripotentdifferentiation potential, thus possessing the capability of developinginto any organ, tissue type or cell type. These cells can be derivedfrom the inner cell mass of the blastocyst, or can be derived from theprimordial germ cells from a post-implantation embryo (embryonal germcells or EG cells). ES and EG cells have been derived from mice, andmore recently also from non-human primates and humans. Evans et al.(1981) Nature 292:154-156; Matsui et al. (1991) Nature 353:750-2;Thomson et al. (1995) Proc. Natl. Acad. Sci. USA. 92:7844-8; Thomson etal. (1998) Science 282:1145-1147; and Shamblott et al. (1998) Proc.Natl. Acad. Sci. USA 95:13726-31.

The terms “stem cells,” “embryonic stem cells,” “adult stem cells,”“progenitor cells” and “progenitor cell populations” are to beunderstood as meaning in accordance with the present invention cellsthat can be derived from any source of adult tissue or organ and canreplicate as undifferentiated or lineage committed cells and have thepotential to differentiate into at least one, preferably multiple, celllineages.

The hepatocytes added to the apparatus of the invention can be highlyproliferative hepatocytes, known as small hepatocytes (SHCs), which havethe ability to proliferate in vitro for long periods of time (Mitaka, etal., Biochem Biophys Res Commun 214, 310 (1995); Taneto, et al, Am JPathol 148, 383 (1996)). Small hepatocytes express hepatocyte specificfunctions such as albumin production (Mitaka, et al., Hepatology 29, 111(1999)).

Methods for Seeding Cells into Molds or Polymer Scaffolds

After the mold with the desired high degree of micromachining isprepared, the molds themselves or polymer scaffolds are seeded with thedesired cells or sets of cells. The distribution of cells throughout themold or polymer scaffold can influence both (1) the development of avascularized network, and (2) the successful integration of the vasculardevice with the host. The approach used in this invention is to providea mechanism for the ordered distribution of cells onto the mold orpolymer scaffold. Cells that are enriched for extracellular matrixmolecules or for peptides that enhance cell adhesion can be used. Cellscan be seeded onto the mold or polymer scaffold in an ordered mannerusing methods known in the art, for example, Teebken, et al., Eur J.Vasa Endovasc. Surg. 19, 381 (2000); Ranucci, et al., Biomaterials 21,783 (2000). Also, tissue-engineered devices can be improved by seedingcells throughout the polymeric scaffolds and allowing the cells toproliferate in vitro for a predetermined amount of time beforeimplantation, using the methods of Burg et al., J. Biomed. Mater. Res51, 642 (2000).

For purposes of this invention, “animal cells” can comprise endothelialcells, parenchymal cells, bone marrow cells, hematopoietic cells, musclecells, osteoblasts, stem cells, mesenchymal cells, stem cells, embryonicstem cells, or fibroblasts. Parenchymal cells can be derived from anyorgan, including heart, liver, pancreas, intestine, brain, kidney,reproductive tissue, lung, muscle, bone marrow or stem cells.

In one embodiment, the mold or polymer scaffold is first seeded with alayer of parenchymal cells, such as hepatocytes or proximal tubulecells. This layer can be maintained in culture for a week or so in orderto obtain a population doubling. It can be maintained in a perfusionbioreactor to ensure adequate oxygen supply to the cells in theinterior. The apparatus is then seeded with a layer of endothelial cellsand cultured further. In regions where the matrix is resorbed rapidly,the tissue can expand and become permeated with capillaries.

Cell Seeding of Horizontal Layer by Laminar Flow

A structure comprising joined or fastened molds and/or polymerscaffolds, with or without a semi-permeable membrane between them, iscalled an “apparatus” for purposes of this invention. Sets of cells canbe added to or seeded into the three-dimensional apparatuses, which canserve as a template for cell adhesion and growth by the added or seededcells. The added or seeded cells can be parenchymal cells, such ashepatocytes or proximal tubule cells. Stem cells can also be used. Asecond set of cells, such as endothelial cells, can be added to orseeded onto the assembled apparatus through other vessels than thoseused to seed the first set of cells. The cell seeding is performed byslow flow. As a practical matter, the geometry of the apparatus willdetermine the flow rates. In general, endothelial cells can enter andform vessel walls in micromachined channels that are about 10-50 μm.Thus, in addition to serving as a mechanical framework for the organ,the assembled apparatus provides a template for all of themicrostructural complexity of the organ, so that cells have a mechanicalmap to locate themselves and form subsystems, such as blood vessels inthe liver.

Optionally, functional cells are seeded into both a first and secondmold and/or polymer scaffold with microchannels on their surfaces, andthe two molds and/or polymer scaffolds are joined or fastened with asemi-permeable membrane between them, allowing gas exchange, diffusionof nutrients, and waste removal. One layer comprises the circulationthrough which blood, plasma or media with appropriate levels of oxygencan be continuously circulated to nourish the second layer. The secondlayer comprises a reservoir for the functional cells of an organ, andoptionally includes inlets for neural innervation, urine flow, biliaryexcretion or other activity. This results in an apparatus for makingtissue lamina, wherein each of the first and second molds and/or polymerscaffolds and the semi-permeable membrane are comprised of material thatis suitable for attachment and culturing of animal cells. The sheet oftissue created by the apparatuses and/or methods of the invention isreferred to as “tissue lamina”.

Channels in the horizontal direction typically proceed from larger tosmaller to larger. The geometries can be as complex as desired in-plane(horizontal direction). Thus, one can use small geometries in-plane(such as horizontal conduits of about 5-20 μm). The alignment ofthrough-holes creates vertical conduits or channels in the z-axis.However, the vertical channels need not go from larger to smaller tolarger. In the vertical direction, the vertical channels are typicallyparallel to each other and have diameters on the micron level, largeenough only to allow cell seeding (e.g., hepatocytes are about 40 μm).In one embodiment, different types of cells are seeded horizontally ontodifferent layers of the assembled apparatus. In another embodiment, thedifferent types of cells are seeded using pores or channels fromdifferent directions.

Although described herein with particular reference to formation ofvascularized tissue, it should be understood that the channels can beused to form lumens for passage of a variety of different fluids, notjust blood, but also bile, lymph, nerves, urine, and other body fluids,and for the guided regeneration or growth of other types of cells,especially nerve cells. The tissue layer can include some lumens forforming vasculature and some for other purposes, or be for one purpose,typically providing a blood supply to carry oxygen and nutrients to andfrom the cells in the tissue.

Molecules such as growth factors or hormones can be covalently attachedto the surface of the molds and/or polymer scaffolds and/orsemi-permeable membrane to effect growth, division, differentiation ormaturation of cells cultured thereon.

EXAMPLES

The Examples presented herein demonstrates that present invention can beadapted to suit the needs of all living tissues. The following examplesare put forth so as to provide those of ordinary skill in the art with acomplete disclosure and description of how to make and use thethree-dimensional systems of the invention, and are not intended tolimit the scope of what the inventors regard as their invention. TheExamples are provided to further describe the invention, and should notbe considered to limit its scope in any way.

Example 1 Micromaching of Template to Tissue Engineer BranchedVascularized Channels for Liver Fabrication

Micromachining technologies were used on silicon and pyrex surfaces togenerate complete vascular systems that can be integrated withengineered tissue before implantation. Trench patterns reminiscent ofbranched architecture of vascular and capillary networks were etchedusing standard photolithographic techniques onto silicon and pyrexsurfaces to serve as templates. Hepatocytes and endothelial cells werecultured and subsequently lifted as single-cell monolayers from thesetwo dimensional molds. Both cell types were viable and proliferative onthese surfaces. In addition, hepatocytes maintained albumin production.

Materials and Methods Micromachining Techniques

Templates for the formation of sheets of living vascularized tissue werefabricated utilizing micromachining technology. For the present work, asingle level etch was utilized to transfer a vascular network patterninto an array of connected trenches in the surface of both silicon andpyrex wafers.

In this prototype, a simple geometry was selected for patterning thevascular network. Near the edge of each wafer, a single inlet or outletwas positioned, with a width of 500 μm. After a short length, the inletand outlet branched into three smaller channels of width 250 μm; each ofthese branched again into three 125 μm channels, and finally down tothree 50 μm channels. Channels extend from the 50 μm channels to form acapillary network, which comprises the bulk of the layout. In betweenthese inlet and outlet networks lies a tiled pattern of diamonds andhexagons forming a capillary bed and filling the entire space betweenthe inlet and outlet. In one configuration, the capillary width was setat 25 μm, while in the other, capillaries were fixed at 10 μm. Thisgeometry was selected because of its simplicity as well as its roughapproximation to the size scales of the branching architecture of theliver. Layout of this network was accomplished using CADENCE software(Cadence, Chelmsford, Mass.) on a Silicon Graphics workstation. A filewith the layout was generated and sent electronically to Align-Rite(Burbank, Calif.), where glass plates with electron-beam-generatedpatterns replicating the layout geometry were produced and returned forlithographic processing.

Starting materials for tissue engineering template fabrication werestandard semiconductor grade silicon wafers (Virginia Semiconductor,Powhatan, Va.), and standard pyrex wafers (Bullen Ultrasonics, Eaton,Ohio) suitable for MEMS processing. Silicon wafers were 100 mm diameterand 525 μm thick, with primary and secondary flats cut into the wafersto signal crystal orientation. Crystal orientation was <100>, and waferswere doped with boron to a resistivity of approximately 5 W-cm. Thefront surface was polished to an optical finish and the back surfaceground to a matte finish. Pyrex wafers were of composition identical toCorning 7740 (Corning Glass Works, Corning N.Y.), and were also 100 mmin diameter, but had a thickness of 775 μm. Both front and back surfaceswere polished to an optical finish. Prior to micromachining, both wafertypes were cleaned in a mixture of 1 part H₂SO₄ to 1 part H₂O₂ for 20minutes at 140° C., rinsed 8 times in deionized water with a resistivityof 18 MW, and dried in a stream of hot N₂ gas.

For silicon and pyrex wafers, standard photolithography was employed asthe etch mask for trench formation. Etching of pyrex wafers requiresdeposition of an intermediate layer for pattern transfer which isimpervious to the etch chemistry. A layer of polysilicon of thickness0.65 μm over the pyrex was utilized for this purpose. This layer wasdeposited using Low Pressure Chemical Vapor Deposition (LPCVD) at 570°C. and 500 mTorr via the standard silane decomposition method. In thecase of silicon, photoresist alone could withstand limited exposure totwo of the three etch chemistries employed. For the third chemistry, a1.0 μm layer of silicon dioxide was thermally deposited at 1100° C. inhydrogen and oxygen.

Once the wafers were cleaned and prepared for processing, images of theprototype branching architecture were translated onto the wafer surfacesusing standard MEMS lithographic techniques. A single layer ofphotoresist (Shipley 1822, MicroChem Corp., Newton, Mass.) was spun ontothe wafer surfaces at 4000 rpm, providing a film thickness ofapproximately 2.4 μm. After baking at 90° C. for 30 minutes, the layerof photoresist was exposed to UV light using a Karl Suss MA6 (SussAmerica, Waterbury, Vt.) mask aligner. Light was passed through thelithographic plate described earlier, which was in physical contact withthe coated wafer. This method replicates the pattern on the plate to anaccuracy of 0.1 μm. Following exposure, wafers were developed in Shipley319 Developer (MicroChem. Corp., Newton, Mass.), and rinsed and dried indeionized water. Finally, wafers were baked at 110° C. for 30 minutes toharden the resist, and exposed to an oxygen plasma with 80 Watts ofpower for 42 seconds to remove traces of resist from open areas.

Silicon wafers were etched using three different chemistries, whilepyrex wafers were processed using only one technique. For pyrex, thelithographic pattern applied to the polysilicon intermediate layer wastransferred using a brief (approximately 1 minute) exposure to SF₆ in areactive-ion-etching plasma system (Surface Technology Systems, Newport,United Kingdom). Photoresist was removed, and the pattern imprinted intothe polysilicon layer was transferred into trenches in the silicon usinga mixture of 2 parts HNO₃ to 1 part HF at room temperature. With an etchrate of 1.7 μm per minute, 20 μm deep trenches were etched into thepyrex wafers in approximately 12 minutes. Since the chemistry isisotropic, as the trenches are etched they become wider. Processing withthe layout pattern with 25 μm wide capillary trenches tended to resultin merging of the channels, while the use of 10 μm wide trenches avoidedthis phenomenon. Interferometric analysis of the channels after etchingshowed that surface roughness was less than 0.25 μm. Once channeletching of pyrex wafers was completed, polysilicon was removed with amixture of 10 parts HNO₃ to 1 part HF at room temperature, and waferswere re-cleaned in 1 part H₂SO₄ to 1 part HF.

Three different chemistries were employed to etch silicon in order toinvestigate the interaction between channel geometry and cell behavior.First, a standard anisotropic plasma etch chemistry, using a mixture ofSF₆ and C4F₈ in a switched process plasma system from STS²⁴, was used toproduce rectangular trenches in silicon. Narrower trenches are shallowerthan deep trenches due to a phenomenon known as RIE lag. A secondprocess utilized a different plasma system from STS, which producesisotropic trenches with a U-shaped profile. While the process isisotropic, widening of the trenches is not as severe as is experiencedin the isotropic pyrex etching process described earlier. In both ofthese plasma etching cases, trenches were etched to a nominal depth of20 μm. For the third process, anisotropic etching in KOH (45% w/w in H₂Oat 88° C.), the intermediate silicon dioxide layer mentioned above wasemployed. First, the silicon dioxide layer was patterned using HFetching at room temperature. The KOH process produces angled sidewallsrather than the rectangular profile or U-shaped profile produced by thefirst two recipes, respectively. Crystal planes in the <111> orientationare revealed along the angled sidewalls, due to anisotropic propertiesof the KOH etch process as a function of crystal orientation. Due to theself-limiting nature of the channels produced by this process, trenchdepth was limited to 10 μm. After completion of the silicon waferetching, all layers of photoresist and silicon dioxide were removed, andwafers were cleaned in 1 part H₂SO₄:1 part H₂O₂ at 140° C., followed byrinsing in deionized water and drying in nitrogen gas.

For this set of experiments, no attempt was made to alter the surfacechemistry of the silicon and pyrex wafers. Prior to processing, siliconwafers were uniformly hydrophobic, while pyrex wafers were equallyhydrophilic, as determined by observations of liquid sheeting andsessile drop formation. After processing, unetched surfaces appeared toretain these characteristics, but the surface chemistry within thechannels was not determined.

Animals

Adult male Lewis rats (Charles River Laboratories, Wilmington, Mass.),weighing 150-200 g, were used as cell donors. Animals were housed in theAnimal Facility of Massachusetts General Hospital in accordance with NIHguide lines for the care of laboratory animals. They were allowed ratchow and water ad libitum and maintained in 12-hour light and dark cycle

Cell Isolations

Male Lewis rats were used as hepatic cell donors. HCs were isolatedusing a modification of the two-step collagenase perfusion procedure aspreviously described by Aiken, et al., J Pediatr Surg 25, 140 (1990) andSeglen, Methods Cell Biol 13, 29 (1976). Briefly, the animals wereanesthetized with Nembutal Sodium Solution (Abbott Laboratories, NorthChicago, Ill.), 50 mg/kg, and the abdomen was prepared in sterilefashion. A midline abdominal incision was made and the infrahepaticinferior vena cava was cannulated with a 16-gauge angiocatheter (BectonDickinson). The portal vein was incised to allow retrograde efflux andthe suprahepatic inferior vena cava was ligated. The perfusion wasperformed at a flow rate of 29 ml/min initially with a calcium-freebuffer solution for 5 to 6 minutes, then with a buffer containingcollagenase type 2 (Worthington Biomedical Corp., Freehold, N.J.) at 37°C. The liver was excised after adequate digestion of the extracellularmatrix and mechanically agitated in William's E medium (Sigma, St.Louis, Mo.) with supplements to produce a single cell suspension. Thesuspension was filtered through a 300 μm mesh and separated into twofractions by centrifugation at 50 g for 2 minutes at 4° C. The pelletcontaining the viable HC fraction was resuspended in William's E mediumand further purified by an isodensity Percoll centrifugation. Theresulting pellet was then resuspended in Hepatocyte Growth Medium, andcell counts and viabilities of HCs were determined using the trypan blueexclusion test.

The endothelial cells were derived from rat lung microvessels and theywere purchased directly from the vendor, Vascular Endothelial CellTechnologies (Rensellaer, N.Y.).

Hepatocyte Culture Medium

William's E medium supplemented with 1 g sodium pyruvate (Sigma, St.Louis, Mo.) and 1% glutamine-penicillin-streptomycin (Gibco BRL,Gaithersburg, Md.) were used during the cell isolation process. Theplating medium was Dulbecco's modified eagle medium (Gibco BRL)supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 44mM sodium-bicarbonate, 20 mM HEPES, 10 mM niacinamide, 30 microgram/mlL-proline, 1 mM ascorbic acid 2 phosphate, 0.1 μM dexamethasone (Sigma),insulin-transferrin-sodium selenite (5 mg/L-5 mg/L-5 μgram/L, RocheMolecular Biomedicals, Indianapolis, Ind.), and 20 ng/mL epidermalgrowth factor (Collaborative Biomedical Products, Bedford, Mass.).

Endothelial Cell Culture Medium

Dulbecco's modified eagle medium (Gibco BRL) was supplemented with 10%fetal bovine serum, 1% penicillin-streptomycin, 25 mg of ascorbic acid(Sigma), 10 mg L-alanine (Sigma), 25 mg L-proline (Sigma), 1.5 microgramcupric sulfate (Sigma), glycine (Sigma) and 1M Hepes buffer solution(Gibco BRL). The media was supplemented with 8 mg of ascorbic acid everyday.

Cell Attachment to Non-Etched Silicon and Pyrex Wafers

Silicon and pyrex were both tested as possible substrates for theculture of endothelial cells and hepatocytes. Prior to cell seeding, thepyrex wafers were sterilized with 70% ethanol (Fisher, Pittsburgh, Pa.)overnight and washed three times with sterile phosphate buffered saline(Gibco BRL). Silicon wafers were first soaked in acetone for 1 hr,followed a methanol rinse for 15 minutes, and overnight sterilization in100% isopropyl alcohol. Rat lung microvascular endothelial cells wascultured on non-coated pyrex and silicon surfaces, as well as waferscoated with vitrogen (30 microgram/ml), Matrigel® (1%), or Gelatin (10mg/ml). Once isolated, the cells were resuspended in endothelial cellculture medium, seeded uniformly onto the wafer at a density of 26.7×10³cells/cm², and cultured at 5% CO₂ and 37° C.

The rat hepatocytes were also cultured on non-coated pyrex and silicon,as well as wafers coated with a thin and thick layers of vitrogen (30microgram/ml and 3 microgram/ml) and Matrigel (1%) in order to determinethe optimal methods for lifting hepatocyte sheets. Once isolated, thehepatocytes were resuspended in hepatocyte growth media, seeded onto thewafer at a density of 111.3×10³ cells/cm², and cultured at 5% CO₂ and37° C. Cell attachment and growth was observed daily using microscopy.

Immunohistochemical Staining

Both membranes were fixed in 10% buffered formalin for 1 hr andharvested for histological study, and the hepatocytes were stainedimmunohistochemically.

The hepatocyte cell monolayer membrane was fixed in 10% bufferedformalin and processed for hematoxylin-eosin and immunohistochemicalstaining using a labeled streptavidin biotin method (LSAB2 kit for ratspecimen, DAKO, Carpinteria, Calif.). The primary antibody was rabbitanti-albumin (ICN, Costa Mesa, Calif.). Three-micron sections wereprepared and deparafinized. The specimens were treated with peroxidaseblocking buffer (DAKO) to prevent the nonspecific staining. Sectionswere stained with albumin diluted with phosphate buffered saline,followed by biotinylated anti-rabbit antibody and HRP conjugatedstreptavidin. Sections were treated with DAB as substrate and werecounterstained with hematoxylin.

Albumin Production

To assess hepatocyte function, albumin concentration in the culturemedium was measured every 24 hours for 5 days pre-cell detachment usingan enzyme linked immunosorbent assay (n=5), as described by Schwere, etal., Clinica Chemica Acta 163, 237 (1987). In brief, a 96 wellmicroplate was coated with anti-rat albumin antibody (ICN). Afterblocking non-specific responses with a 1% gelatin solution, each samplewas seeded onto the plate and incubated for 1 hour at 37° C. This wasfollowed by another 1 hour incubation with peroxidase conjugatedanti-rat albumin antibody (ICN). Finally, the substrate was added andextinction was measured with a microplate reader at 410 nm. R² of thestandard curve was >0.99. Results demonstrate continued production ofalbumin by cultured hepatocytes (FIG. 10).

Statistical Analysis

All data was expressed as mean±SD. Statistical analysis was performedwith a paired t-test. Statistical significance was determined as whenthe p value of each test was less than 0.05.

Cell Attachment to Etched Silicon and Pyrex Wafers

Endothelial cells and hepatocytes were also seeded onto etched siliconand pyrex wafers. Prior to cell seeding, the pyrex wafers weresterilized with 70% ethanol (Fisher) overnight and washed three timeswith sterile phosphate buffered saline (Gibco BRL). Silicon wafers werefirst soaked in acetone for 1 hr, followed a methanol rinse for 15minutes, and overnight sterilization in 100% isopropyl alcohol. Ontothese wafers were seeded rat lung microvascular endothelial cells at adensity of 26.7×10³ cells/cm², or rat hepatocytes at a density of111.3×10³ cells/cm². These cells were cultured at 5% CO₂ and 37° C., andtheir attachment and growth observed daily using microscopy.

Results Micromachining

A schematic of the vascular branching network design used as a templatefor micromachining is shown in FIG. 11A. This pattern was transferred tosilicon and pyrex wafers using the processes described in the Materialsand Methods section. Typical trench depths of 20 μm on silicon and 10 μmon glass were achieved utilizing these processes. An optical micrographof a portion of the capillary network etched into a silicon wafer isshown in FIG. 11B. In FIG. 11C, a Scanning Electron Micrographcross-section of an angled trench etched using the anisotropic etchingprocess described earlier is shown. This process resulted in excellentadhesion and enhanced lifting of living tissue.

Growth of Cells on Silicon and Pyrex Wafers

The adhesion and growth of endothelial cells and hepatocytes on severaldifferent substrate surfaces were compared. On all pyrex wafers, coatedor non-coated, the endothelial cells proliferated and grew to confluencewithin four days. Hepatocytes also attached and spread well on allcoated and non-coated pyrex wafers. Histological assessment of thedetached cell monolayers of both hepatocytes and endothelial cellsmanifested promising results. Hemotoxylin and Eosin (H&E) staining ofboth showed that all cells were viable and that most were undergoingmitoses. The endothelial cells were observed to be primarily attenuatedand to form a single-celled alignment. The monolayer of hepatocytesshowed each cell to be of a spheroid configuration with eosinophilicflocculent cytoplasm and a large nucleus with a bright red nucleolus,similar to that seen in the native liver. Moreover, cellular attachmentswere less attenuated than the endothelial cells. Thus, these results arereminiscent of each of the cell types' specific functions. In biologicalsystems, the endothelium functions to provide a thin, smooth outersurface of a barrier and a transport channel and so it is understandablethat these cells are observed here to be primarily attenuated and in asingle-celled array. The hepatocytes have more of a tendency to formtissue and so less of a single-celled array and more of a roundedmulti-layered array is seen.

Albumin secretion into the hepatocyte culture medium at day 2, 3, 4, and5 was 165.96±29.87, 164.44±17.22, 154.33±18.46, 115.47±18.09(microgram/day, Graph 1), respectively. Though there was a statisticallysignificant difference between day 4 and day 5, no significantdifferences were observed between day 2, day 3, and day 4 (p<0.05 by thepaired t-test). Hence, this data shows that cells cultured on siliconwafers were able to maintain a fairly constant albumin production rateuntil day 4.

Moreover, through immunohistochemical staining of the detachedhepatocyte monolayers, many cells were stained positive for albuminindicating further that hepatocyte function was maintained on siliconwafers.

Implantation of Hepatocyte Sheet into the Rat Omentum

H&E staining of hepatocyte sheets implanted into rat omentumdemonstrated that all cells were viable and showed proliferation at fourweeks and three months. The implanted hepatocyte monolayer sheets, whenharvested, were over 5 cell layers thick in most areas.

This study demonstrates that silicon microfabrication technology can beutilized to form large sheets of living tissue. It also demonstrates thefeasibility of etching ordered branching arrays of channels that allowliving endothelial cells to line the luminal surface of the channels. Inaddition, it has been shown that organized sheets of engineeredhepatocyte tissue and endothelial tissue can be lifted from the surfaceof silicon or pyrex wafers and can be folded into a compactthree-dimensional configuration. The hepatocyte sheets have then beenplaced into rats on the highly vascular surface of the omentum. Thatstructure has then been rolled into a three-dimensional cylinder as amodel for an engineered vasculature. Vascularized hepatic tissue wasformed as a permanent graft.

Example 2 Endothelialized Microvascular Networks Grown on MicromachinedPyrex® Templates for Tissue Engineering of Vital Organs

This Example shows the design, modeling, and experimental/computationaltesting of the endothelialized microvascular matrices grown onmicromachined Pyrex® templates.

Patterns of microvascular networks were etched using microfabricationtechnologies on Pyrex® wafers. The pattern consisted of 10 generationsof bifurcations from a single inflow channel of width 3 mm down tochannels of width of 30 μm.

The channels were then collected to a single outflow. All channels wereetched to the same depth of 30 μm. The Pyrex® wafer was sealed to a flatsilicone rubber sheet of the same size. Endothelial cells harvested fromrat lung were successfully seeded and expanded under continuous flowconditions in this microvascular network. Red blood cells harvested fromrat were heparinized and perfused into the endothelialized channels, andsuccessfully collected at the output. Using micro-visualizationtechniques, the concentration of red blood cells (hematocrit) in themicrovascular network was measured. The distribution of blood flow rate,pressure, and hematocrit was also calculated in the entire microvascularsystem using an earlier developed computational algorithm.

Epithelial cells were observed flowing through channels and attachingmainly around the walls of smallest channels on day 1 and growing toconfluence along the channels under continuous flow conditions over thefollowing 5 days. Rat lung endothelial cells attach in a single layer tothe walls of these mold structures without occluding them.

Hematocrit compared well between the experimental measurements andnumerical calculations. Red blood cells reach even the smallest vesselsin the network, ensuring sustained transport of oxygen to the engineeredcapillaries.

In summary, microfabrication technology is demonstrated as an approachfor organizing endothelial cells in vitro at the size scale of themicrocirculation.

Example 3 Microfluidics Device for Tissue Engineering MicrovasculatureEndothelial Cell Culture

In this Example, the fabrication of the microfluidic mold, in vitroseeding, and extended cell culture in the mold is demonstrated.Capillary networks were fabricated in biocompatible PDMS, sterilized,coated with cell adhesion molecules, and seeded with cells.Cell-containing molds were then connected to a closed-loop bioreactorfor long-term culture. Continuous-flow culture of endothelial cells forup to 4 weeks without occlusion or contamination was achieved.

Traditional soft lithography microfluidics were used as a prototypematrix. These cell-containing microfluidics are capable of supportinglong-term culture in vitro, because in vitro expansion of cells prior toimplantation can take several weeks. The prototype matrix is designed tosupply sufficient oxygen and nutrients and to remove excretory productswhile avoiding large shear stresses. The matrix is useful for long-termmicrofluidic cell culture, including the maintenance of sterility andthe minimization of cell and bubble occlusions.

Microfluidic networks that support physiologic flows and pressures weredeveloped by photopatterning SU-8, a high-aspect ratio negativephotoresist, onto silicon. This was used as a mold for castingpolydimethylsiloxane (PDMS). After removal from the mold, inlets andoutlets were cored with blunted syringe needles, and the micropatternedpolymer scaffold was irreversibly sealed to an unpatterned layer ofPyrex® or PDMS by oxygen plasma surface treatment. See Duffy, et al.,Anal. Chem. 70, 4974 (1998). The microfluidic device was autoclavesterilized and perfused with a solution containing cell adhesionmolecules (poly-L-lysine, collagen, gelatin, or fibronectin), which wereallowed to adsorb for one hour.

The fluidic network was then seeded with a 1×10⁶-1×10⁸ cells/mL cellsuspension using a syringe pump at flow rates ranging from 10-100μL/min. The cells were then allowed to attach for 24 hours, after whichthe device was connected in-line with a sterile bioreactor consisting ofa peristaltic pump, oxygenator, bubble trap, and a reservoir of sterileculture medium. Sterile culture medium was pumped peristaltically from asterile reservoir through an oxygenator consisting of along length oftubing semipermeable to oxygen. The oxygenator was followed by a smallbubble trap, leading directly to the microfluidic circuit. Finally, thesystem was run closed-loop in an incubator at standard culture settings.

Autoclave sterilization of the microfluidic circuit caused no obviouspattern distortion. Coating the channels with cell adhesion moleculesenhanced cell attachment when compared to phosphate bufferedsaline-coated control channels. Seeding of cells into channels of widthsbetween 30-200 mm was optimized by varying concentrations and flowrates. The continuous-flow bioreactor was used to dynamically cultureendothelial cells at flow rates between 0.01 mL/min and 0.1 mL/min. Bothsingle channels and complex networks of channels (30-200 μm wide and 40μm deep) were successfully seeded and cultured. In 100 μm×40 μm singlechannels, cells were cultured for more than 4 weeks withoutcontamination or occlusion.

Long-term culture of cells in microfluidic devices was achieved. Cellssuccessfully attached, proliferated, and migrated in closedmicrofabricated channels with small geometries.

Example 4 Generation of Functionally Differentiated, Three-DimensionalHepatic Tissue from Two-Dimensional Sheets of Small Hepatocytes andNon-Parenchymal Cells

In this Example, three-dimensional, vascularized liver tissue wasfabricated in vivo from a non-vascularized monolayer or cell sheet ofsmall hepatocytes (SHCs) formed on a silicon wafer. SHCs cells aresmaller than mature hepatocytes (MHCs), but morphologically similar,with a highly proliferative capacity (Mitaka, et al., Biochem BiophysRes Commun 214, 310 (1995); Mitaka, et al., Gastroenterol Hepatol 13Suppl, S70 (1998); Mitaka, et al., Hepatology 29, 111 (1999); Tateno, etal., Am J Pathol 148, 383 (1996); Tateno, et al., Am J Pathol 149, 1593(1996)).

Cell sheets created from SHCs and NPCs were implanted onto rat omentumwith maximal hepatotrophic stimulation by retrorsine, portacaval shunt,and partial hepatectomy, and their engraftment and function wereevaluated. Using this cell type, co-cultured with non-parenchymal cells(NPCs), liver tissue that maintained a high level of albumin productionwas fabricated in a flow culture system. Animals as described in Example1 were used as cell donors. Cells were cultured as described in theHepatocyte Culture Medium section of Example 1.

Cell Isolation

SHCs and NPCs were isolated by the process described in Example 1, withthe following modifications. Animals were anesthetized by anintramuscular injection with Ketamine and Xylazine. Cells werecollected, suspended, filtered and centrifuged as previously described.Following centrifugation, the pellet containing a majority of MHCs wasdiscarded. The supernatant was collected, and the fraction containingSHCs and NPCs was obtained as a pellet by additional centrifugationtwice at 150×g for 5 minutes. The pellet was resuspended in the platingmedium and the cell number and viability were counted using the trypanblue exclusion test.

In Vitro SHC Sheets Preparation

In order to obtain SHC sheets, the SHCs and NPCs were seeded andcultured on silicon wafers (10 cm diameter). Briefly, the silicon waferswere sterilized with ethylene oxide gas and coated with liquid collagen(Vitrogen 100, Collagen Corp., Palo Alto, Calif.). The mixture of SHCsand NPCs was resuspended in the plating medium at a density of 0.8×10⁶cells/mL. A 25 mL suspension was seeded onto the silicon wafer in a 15cm Petri dish and incubated at 37° C., 5% CO₂. The plating medium waschanged every other day.

Albumin Production

To assess SHC function before implantation, albumin concentration in theplating medium was measured at 3, 5, 7 and 10 days after cell seedingusing an enzyme linked immunosorbent assay (ELISA) (n=11) as describedin Example 1.

In Vivo Model

Retrorsine was administered into the peritoneal cavity of recipient rats(n=23) at a dose of 3 mg/ml/100 g on day 0, and after two weeks aspreviously reported (Laconi, et al., Am J. Pathol 153, 319 (1998)).Three weeks after the second administration, an end-to-side portacavalshunt was created using 8-0 Ethilon suture (ETHICON, Somerville, N.J.)to generate systemic hepatotrophic stimulation for SHC sheetimplantation. One week later, a SHC sheet was spread onto the ratomentum and rolled from distal to proximal into a three-dimensionalcylinder. The omentum was sutured to the anterior wall of the stomachusing 7-0 Prolene suture (ETHICON). A 60% partial hepatectomy wasperformed simultaneously for hepatotrophic stimulation. Animals weresacrificed at the designated time points after SHC sheet implantationfor specimen retrieval. The resected specimens were fixed in 10%formalin solution (Sigma), routinely processed and embedded in paraffinfor subsequent hematoxylin-eosin (H & E) and immunohistochemicalstaining. Two specimens were fixed in 2.5% gluteraldehyde (Sigma) forelectron microscopy (EM).

Immunohistochemical Staining

To characterize the implanted constructs, immunohistochemical stainingusing the Avidin-biotin peroxidase complex (ABC) method was performed.The primary antibodies included: rabbit anti-albumin (DAKO, Carpinteria,Calif.), rabbit anti-transferrin (ICN), mouse anti-pancytokeratin(Sigma), goat anti-γ-glutamyl transpeptidase (GTT) (a gift from Dr.Petersen, Department of Pathology, University of Florida, FL). Four μmparaffin sections were deparaffinized and treated with 4.5% H₂O₂ inmethanol. The specimens were digested for 12 minutes with 0.1% trypsinsolution, followed by treatment with avidin D (Vector) and 5% serum.Subsequently, slides were incubated with the respective primary antibodythat were diluted in phosphate buffered saline with 1% bovine serumalbumin overnight at 4° C. Biotinylated anti-mouse/rabbit/goat antibodywas used as a secondary antibody in combination with the Vectastain ABCkit (Vector, Burlingame, Calif.). Finally, specimens were treated with3-amino-9-ethylcarbazole (AEC) (Vector) as substrate and werecounterstained with Mayer's hematoxylin solution (Sigma).

Electron Microscopy (EM)

Two rats at 4 months were sacrificed for EM study. Immediately afterremoval from the animal, 1 mm sections were placed into Karnovsky's KIIsolution (2.5% glutaraldehyde, 2.0% paraformaldehyde, 0.025% calciumchloride, in a 0.1 M sodium cacodylate buffer, pH 7.4), fixed overnightat 4° C., and routinely processed for EM. Representative areas werechosen from 1 μm sections stained with toluidine blue. The sections wereexamined using a Phillips 301 transmission electron microscope.

Morphologic and Quantitative Analysis

For morphologic and quantitative analysis, specimens were harvested at 2weeks (n=7), 1 month (n=7), and 2 months (n=7). At each time point, therolled omentum was cut perpendicularly to the greater curvature ofstomach, and three to four cross-sections of tissue were obtained andstained with H & E. The area occupied by implanted constructs in eachsection was measured using computer assisted analysis with NIH Imageversion 1.61 software (Division of Computer Research and Technology,NIH, Bethesda, Md., USA). This was expressed as μm²/section.

Statistical Analysis

All values are expressed as mean f SD and were statistically evaluatedusing the Mann-Whitney test or the paired t-test. A value of p<0.05 wasconsidered statistically significant.

Cell Isolation and Growth in a Culture Flask

All cell isolations yielded 8-14×10⁷ cells comprising SHCs and NPCswith >90% overall viability. To evaluate the culture condition of SHCson silicon wafers, a cell suspension was seeded on culture flasks at thesame concentration. One day after seeding, most cells began to attachindividually or occasionally form small clusters consisting of severalSHCs. After 3 days, cells have completely attached and spread on theculture flask. After 5 days, many clusters had formed and NPCs wereobserved between the clusters. These small clusters united to formlarger clusters and continued to grow until implantation (FIG. 12).

Cell Sheet Formation

SHCs and NPCs cultured on silicon wafers grew similarly to the cultureflask. Many large clusters were observed macroscopically on the siliconwafers after culturing for 10-14 days. Cultured cells were lifted as asheet from all the silicon wafers. After lifting, cell sheets shrunk toapproximately 2.5 cm in diameter (FIG. 13).

Albumin Production

Albumin secretion at day 3, 5, 7, and 10 was, 6.47±2.49, 12.08±5.18,19.93±4.05, 30.14±5.46 (μg/day), respectively (FIG. 14). There werestatistically significant differences between day 3 and day 5, day 5 andday 7, and day 7 and day 10 (p<0.05 by the paired t-test).

H & E Staining

The H & E staining of specimens harvested at 2 weeks after cell sheetimplantation typically reveal large, polygonal, eosinophilic cells withround nuclei resembling hepatocytes, cuboidal cells resembling biliaryepithelial cells, and capillary formation. At this time point the areaof hepatocytes was less than five cell layers thick (FIG. 15A). At 1 and2 months, large clusters of hepatocytes over five cell layers thick,cuboidal cells resembling biliary epithelial cells, and capillaryformation could be observed (FIG. 15B-D). In some areas, hepatocytesexceeded ten cells layer thick. In the specimens at 2 weeks and 1 month,there were many areas that were occupied mainly by bile duct-likestructures (FIG. 15D). As the implant matured in the omentum, the numberof hepatocytes increased and the number of bile duct-like structuresdecreased at 2 months.

Immunohistochemistry

Both hepatocytes and bile duct-like structures stained positively withpan-cytokeratin. However, bile ductules stained more strongly positivethan hepatocytes (FIG. 16A). Since there are normally no pan-cytokeratinpositive cells in the omentum, it is likely that the cells originatedfrom the implanted constructs. Some of the hepatocytes stainedpositively for albumin and transferrin, which suggests that theycontinued to express liver specific functions. The bile duct-likestructures stained positively for GGT, an enzyme expressed at highlevels in normal rat intrahepatic biliary epithelial cells but typicallynot detected in normal rat hepatocytes, and negatively for albumin andtransferrin, which indicated that they were composed of cells resemblingnormal biliary epithelial cells (FIG. 16B-D).

In one case, histology showed that one bile duct-like structure at 2weeks was formed with both cells resembling biliary epithelial and cellswhich were morphologically more similar to hepatocytes (FIG. 17). Thisbile duct-like structure was located between the canaliculi-likestructures composed of hepatocytes, and the bile duct-like structuresformed solely by cells resembling biliary epithelial as if it were atransitional structure between the two. This phenomenon demonstratesthat canaliculi-like structures and bile duct-like structures grow toconfluence in tissue engineered constructs.

Ultrastructure of the Implanted Construct

TEM revealed that the engineered constructs were composed of cells withlarge round nuclei, numerous mitochondria and peroxisomes, andmicrovilli; characteristic of hepatocytes. These cells formed structuresresembling bile canaliculi at the cell-cell borders. Capillaries wereseen between hepatocytes (FIG. 18B).

Morphologic and Quantitative Analysis

The calculated areas occupying implanted constructs were 43136±36181,153810±106422, and 224332±142143 μm²/section at 2 weeks, 1 month, and 2months, respectively. The mean area increased over time, and there weresignificant differences between 2 w and 1 m (p<0.05), and between 2 wand 2 m (p<0.01). No significant difference was observed between 1 m and2 m. The areas occupied by bile duct-like structures were 13407±16984,15430±8980, and 1290±2052 μm²/section at 2 weeks, 1 month, and 2 months,respectively. The areas were significantly greater at 2 weeks (p<0.01)and 1 month (p<0.05 by the Mann-Whitney U test), compared to the area at2 months (FIG. 19).

This Example shows morphologically simple cell sheets created from SHCsand NPCs implanted and engrafted in the omentum. Given adequatehepatotrophic stimulation, implants formed morphologically complexthree-dimensional tissue consisting of hepatocytes, structuresresembling bile canaliculi, and ducts composed of cells resemblingbiliary epithelium. These results represent a significant advance towardthe tissue engineering of complex vascularized thick tissues.

Example 5 Generation of an Ex Vivo Renal Device

This Example describes a microfabricated network of proximal tubulesthat could conduct the essential reabsorptive and excretory functions ofthe kidney ex vivo. (See FIGS. 20-23.) A glomerular endothelialcell-lined network can provide filtration while minimizing thrombosis.These two networks combined on bioresorbable polymer are the basis foran ex vivo tissue engineered renal device.

The design of an ex vivo tissue engineered system can be focused on thedevelopment of a glomerular endothelial filter in conjunction with aproximal tubule device for reabsorption and excretion. The endothelialfilter is specifically designed to provide physiologic flow with lowthrombogenicity and maximized surface area for solute transport. Theproximal tubule device, containing an appropriate number of cells forrenal replacement, has optimized surface area for solute reabsorptionand an outlet for urine excretion (See FIG. 32). Several layers of moldsand/or polymer scaffolds and semi-permeable membranes can be stacked tooptimize filtration and reabsorption. Biocompatible, bioresorbable andmicroporous polymers are used throughout for optimal cell growth andfunction.

Materials and Methods Configuring the Mold

MEMS replica molding was used to create the polymer molds used in thisExample. Using the techniques described herein, an inverse pattern(i.e., protrusions rather than indentations) corresponding to thedesired pattern of microchannels was formed on a silicon wafer.Poly-(dimethyl siloxane) (PDMS) was then cast onto the silicon template.After the template was removed, the PDMS was subjected to O₂ plasmatreatment, and was fastened to a second layer of PDMS. In this Example,the second layer of PDMS was flat, however, in other embodiments, eitheror both surfaces of the second PDMS layer can contain a pattern ofmicrochannels. In addition, a semi-permeable membrane can be fastenedbetween the PDMS layers.

Cell Culture

Renal proximal tubule cells and glomerular endothelial cells from ratand pig models have been isolated using sieve filtration and separationover a Percoll gradient (Vinay, et al. Am J Physiol 241, F403 (1981);Misra, et al. Am J Clin Path 58, 135 (1972)). Human microvascularendothelial cells were isolated from normal neonatal foreskin incollaboration with Dr. Michael Detmar (Cutaneous Biology ResearchCenter, MGH Charletown), and stained positively for endothelial cellmarkers CD-31 and von Willebrand's factor (vWF) within the PDMS devices.

Both renal proximal tubule cells and human microvascular endothelialcells were seeded into the MEMS-designed PDMS (poly(dimethyl siloxane))devices at 20 million cells/ml. Cells were allowed to adhere at 37° C.for six hours. Devices were rotated 180 degrees at three hours to allowadherence of cells to both sides of the microchannels. Flow was thenstarted via infusion pump with appropriate culture medium to maintaincell viability.

Results

Human microvascular endothelial cells were seeded into poly(dimethylsiloxane) (PDMS) microchannels (smallest channel width 30 μm, depth 35μm) using the specifically designed MEMS templates, and good celladherence and proliferation within the channels was observed (FIG. 24).

A computational model is used to maximize blood flow through theglomerular cell filter, within normal hemodynamic parameters. FiniteElement Modeling (FEM) technologies are used to maximize the surfacearea for filtration to simulate mass transport of solutes across thefilter. The template topography and branching angles are designed tominimize thrombosis within the microchannels. Similarly, the proximaltubule network is optimized to provide even flow distribution, surfacearea for reabsorption, and an outflow tract for excretion of urine.

Cultured proximal tubular cells exhibit characteristic dome formation.Glomerular endothelial cells have also been isolated and maintained inculture. Further characterization of the cells is performed usingimmunohistochemical staining. Proximal tubule cells are stained formegalin (gp330) expression, and endothelial cells are stained for vonWillebrand's factor (vWF) and CD-31.

Function of proximal tubule cells is assessed with the conversion of1,25-OH-D₃ to 1,25-(OH)₂D₃ (1,25-dihydroxyvitamin D₃), the reclamationof glutathione and the generation of ammonium using a single passperfusion system. 25-(OH) D3-12-hydroxylase is a cytochrome P-450monooxygenase found in the inner mitochondrial membrane of proximaltubule cells. Proximal tubule glutathione reclamation is performed bythe brush-border enzyme gamma-glutamyl transpeptidase. In addition,specific transport functions such as vectoral fluid transport (inhibitedby ouabain, an Na⁺—K⁺-ATPase inhibitor), active bicarbonate and glucosetransport (inhibited by acetazolamide and phlorizin respectively), andpara-aminohippurate secretion (inhibited by probenecid) are also tested(Humes, et al., Kid Int 55, 2502 (1999); Humes, et al. Nat Biotechnol17, 451 (1999)). Glomerular endothelial cell function is assessed forpermeability to water and serum proteins, and the basement membranecomponents analyzed.

Microvascular endothelial and proximal tubule cells into have beensuccessfully seeded into PDMS networks made from MEMS templates. FIGS.25-27 show proximal tubule cells growing in the microchannels of thepolymer scaffold at various intervals after seeding.

Example 6 Drug Metabolism in Three-Dimensional Liver Tissue EngineeredSystems Generation of Vascular and Hepatic Tissues on MicrofabricatedSubstrates

HepG2/C3a cells were suspended in modified Eagle's medium α (MEMα),supplemented with 10% fetal bovine serum (no other supplements wereadded). The cells were seeded into the parenchymal chamber of amicrofabricated device at 2 million cells per ml, or 0.5 million cellsper device. The opposite side of the chamber comprised the engineeredvascular tissue. Cultures were maintained at 37° C. FIG. 43A depicts aschematic of the single-pass flow microfabricated device and thephotographs of FIGS. 43B and 44A show HepG2/C3a cells seeded into thedevice. This single-pass flow microfabricated polydimethylsiloxane(“PDMS”) device allows for fresh media to pass through the vasculaturechannels, allowing for nutrient, waste and gas exchange to occur througha polycarbonate membrane (“PC”) separating the parenchymal chamber. Toinitialize the device prior to cell seeding, a 30 mL syringe was filledwith media and placed on a Harvard Apparatus PHD 2000 Infusion pump. Thesyringe was connected by luer locks to silastic tubing, which connectsto the inlet of the microvascular side on the device. The outlet of themicrofabricated channels were connected to another length of silastictubing that is connected to a 22-gauge needle. The needle was insertedinto a bug stopper and bottle where effluent media was collected.

HepG2/C3a cell viability was determined using the LIVE/DEAD® stain(Molecular Probes, Eugene, Oreg.). FIG. 44B shows the appearance ofhealthy hepatocytes under normal culture conditions. These viabilityassays have been carried out to two weeks to demonstrate HepG2/C3asurvival in the MEMS system (FIG. 45).

DNA Content of Human Hepatocyte Cells

HepG2/C3a cells were assayed for DNA content after growth in themicrofabricated device for a total of 10 days. The DNA levels ofHepG2/C3a cells increased up to day 10. The DNA levels were determinedusing the Dneasy Kit from Qiagen at day 1, 3, 5, 7, and 10. The cellswere lysed in the parenchymal chamber of the device using 200 μL of PBS,20 μL of Proteinase K, and 200 μL Buffer AL while placed in a 70° C.water bath. After 10 minutes the devices were removed and 200 μL of 200proof ethanol was added. The solution was then flushed into spin columnsand centrifuged for 1 minute at 8,000 RPM. The collection tube wasdiscarded, 500 μL of Buffer AW1 was added to the column, and thencentrifuged for 1 minute at 8,000 RPM. Again the collection tube wasdiscarded, 500 μL of Buffer AW2 was added to the column, and thencentrifuged for 3 minutes at full speed. The column was placed in aclean microcentrifuge tube and 200 μL of Buffer AE was pipeted onto themembrane. The column was incubated at room temperature for one minuteand centrifuged at 8,000 RPM. The resulting elutant was analyzed for DNAcontent using ultra violet spectrophotometry at wavelengths 260 nm and280 nm. DNA standard regression curves were compiled using the sameprotocol and known number of HepG2/C3a cells.

Liver Cell Function

Liver function was assessed by measuring the production of albumin,alphafetoprotein and transferrin by ELISA. Serum-free HepatocyteMaintenance Media (“HMM”) was recovered from the device on day 1, 3, 5,7 and 10. At each time point, 100 μl was taken and analyzed for thepresence of the liver markers by Enzyme-Linked Immunosorbent Assay(ELISA). The ELISA was performed in a 96 well plate coated with 100 μLof 11.2 μg/mL of unconjugated Rabbit anti-human Albumin by Dako dilutedin a bicarbonate buffer coating solution (0.159 g Sodium CarbonateAnhydrous, 0.0293 g Sodium Bicarbonate, 0.02 g Sodium Azide, 100 mLdistilled water, pH 9.6) and refrigerated overnight. The plates werewashed three times using PBS-Tween (16 g NaCl, 0.04 g Na H₂PO₄monobasic, 2.82 g NaH₂PO₄ sodium monobasic, 0.4 g KCl, 1 mL PolyoxyEthylene Sorbitan, 2 L Distilled Water, pH 7.4) and blocked with 200 μL1% Gelatin Blocking Solution in PBS-Tween. Beginning with 200 μL, eightserial dilutions decreasing the concentration by half, but keeping atotal of 200 μL were made of both the bioreactor sample and the standard25 ηg/mL Human Albumin (Pierce). The plate was incubated for one hourand then washed three times with PBS-Tween. The prepared conjugatedantibody was Dako rabbit anti-human Albumin conjugated to HRP. 50 μL ofthe stock conjugated antibody was diluted into 950 μL of PBS-Tween. 900μL of the resulting solution was diluted into 22 mL of PBS-Tween; 100 μLof this solution was added to each well and incubated at 37° C. for onehour. The wells were washed three times with PBS-Tween and 100 μL ofsubstrate solution was added to each well. The absorbance was measuredat 405 ηm in a Wallac Victor² 1420 MultiLabel Counter plate reader. Theabsorbance was recorded and the measurement of albumin synthesis wascalculated in mcg/dL of media.

ELISA kits for human alphafetoprotein, AFP ELISA Kit (catalog#0500-AFP), were purchased from Alpha Diagnostic (San Antonio, Tex.).Protocol was followed per Alpha Diagnositc Lab's instructions. In brief,96 well strip plates were supplied by Alpha Diagnostic labs, which werepre-coated with primary antibody and ready-to use. One hundredmicroliter of samples were then placed into each well of the first rowfor serial dilution and in duplicates. They were incubated at roomtemperature for 30 minutes before washing 5 times with tap water. Onehundred microliter of secondary antibody linked to HRP enzyme conjugatewas then applied for 30 minutes at room temperature and washed 5 timeswith tap water. Finally, HRP-substrate solution was applied and allowedto react for 10 minutes in the dark at room temperature before applying50 μl of stop solution. All plates were read by the Wallac Victor² 1420MultiLabel Counter plate reader at 450 nm.

ELISA kit for human transferrin (catalog #E80-129) was purchased fromBethyl Labs (Montgomery, Tex.). Protocol per Bethyl Labs was followed.In brief, primary anti-human transferring antibody supplied by BethylLabs was used to coat 96 well plates (Nunc) for 60 minutes, washed 3times with wash solution, blocked for 30 minutes with blocking solution,and washed again for 3 times. After this, the plate was used forstandards and sample testing. One hundred microliter of sample orstandard was placed in each well in duplicates and for serial dilution.As per protocol a 1:100,000 dilution of the HRP conjugate and diluentwas placed in each well and allowed to incubate for 60 minutes. Afterthe incubation step, the wells were washed 5 times. One hundredmicroliter of TMB ELISA solution was placed into each well and allowedto react for 30 minutes after which Stop Solution [2M H₂SO₄] was addedto each well. The plate was then read by the Wallac Victor² 1420MultiLabel Counter plate reader at 450 nm.

Metabolism of 7-ethoxycoumarin

The hepatocyte culture medium was changed from MEMα to HepatocyteMaintenance Medium (Clonetics™), which comprised 50 UL of Dexamethasone(0.5 mL), 57 UL Insulin, Bovine (0.5 mL), and 0.5 mL Gentamicin SulphateAmphotericin-B (0.5 mL) (Single Quots® by Clonetics™). Hepatocytemaintenance medium (HMM) does not contain serum. For the purposes ofmeasuring metabolic function of the hepatocyte cell cultures, thehepatocyte culture medium was additionally supplemented with 35 μM7-ethoxycoumarin (ECOD). The ECOD/HMM was allowed to perfuse through thedevice for 48 hours.

The HMM was not only assayed for the presence of liver cell markers, butalso ECOD and its metabolites. A Supelcosil LC-8 high performance liquidchromatography (HPLC) column (5 μm; 5×0.46 cm) was used to detect ECODand breakdown products thereof. The flow rate was set at 1 ml/min, andthe products detected in the ultraviolet range, at 325 nm. The total runtime was 20 minutes, using a two-solvent gradient. Solvent A wascomprised of 5 ml 1M tetrabutyl ammonium dihydrogen phosphate (TBAP),2.25 ml glacial acetic acid, 900 ml of deionized water, and HPLC-gradeacetonitrile to a final volume of 1000 ml. The solution was adjusted topH 4.7 using NaOH. Solvent B comprised 5 ml of 1M TBAP, 1.25 ml glacialacetic acid, 500 ml water, and acetonitrile to a final volume of 1000ml. Solvent B was adjusted to pH 4.7 using NaOH. Table 1 presents thepercentage of each solvent present in the gradient, correlating to therun time of the HPLC program.

TABLE 1 Time Percentage of Solvent (minutes) Solvent A Solvent B 0 90 104 90 10 12 50 50 12.5 90 10 15 90 10

Four standards were used: 7-ethoxycoumarin, which was prepared as 1mg/ml solution in methanol, and 10 μg/ml solutions prepared from the 1mg/ml solutions in distilled water; the Phase I metabolite of ECOD,7-hydroxycoumarin (7-HC), which was prepared as a 1 mg/ml solution in50:50 methanol:water, and 10 μg/ml solutions diluted in distilled water;the Phase II metabolite, 7-HC-glucuronide, which was prepared as a 1mg/ml solution in 50:50 methanol:water, and subsequently diluted indistilled water to yield a 10 μg/ml solution; and 7-HC-sulphate,prepared as a 1 mg/ml solution in 50:50 methanol:water, and subsequentlydiluted in distilled water to yield a 10 μg/ml solution. ECOD elutesbetween 12.919 to 14.647 minutes from the column, while 7-HC elutes at3.709 to 4.668 minutes. The subsequent breakdown product,7HC-glucuronide, is retained at 2.265 to 3.947 minutes, and 7HC-sulphateis retained from 10.230 to 12.878 minutes.

FIG. 46 depicts a graph of the amount of ECOD and its subsequentbreakdown products in picomoles per cell after 1, 3, 5, 7, and 10 daysof incubation in the microfabricated device. A related graph shown inFIG. 47 depicts the same data in terms of total micromoles of ECOD orits metabolites (FIG. 46 depicts the metabolism on a cellular basis andFIG. 47 depicts the metabolism occurring in the device as a whole).FIGS. 48-51 depict graphs showing each individual metabolite alone onthe same time scale.

The results indicate that three-dimensional liver tissue engineeredsystems metabolize drugs that use Phase I and Phase II cytochrome P450(CYP450) pathways.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theappended claims is not to be limited to particular details set forth inthe above description, as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.Modifications and variations of the method and apparatuses describedherein will be obvious to those skilled in the art, and are intended tobe encompassed by the following claims.

1. A method for determining metabolism of a test agent in a tissuecomprising: A) incubating a test agent and an enzyme within athree-dimensional tissue engineered system, such that anenzyme-substrate complex is formed between the enzyme and the testagent; and B) detecting one or more metabolites of the test agent. 2.The method of claim 1, wherein the tissue-engineered system comprisesliver tissue, kidney tissue, cardiac tissue, cartilage tissue, or bonemarrow tissue, and combinations thereof.
 3. The method of claim 1,wherein the test agent comprises a compound selected from the groupconsisting of opioid analgesics, anti-inflammatory drugs such asantihistamines and non-steroidal anti-inflammatory drugs (NSAIDs),diuretics such as carbonic anhydrase inhibitors, loop diuretics,high-ceiling diuretics, thiazide and thiazide-like agents, andpotassium-sparing diuretics, agents that impinge on the renal andcardiovascular systems such as angiotensin converting enzyme inhibitors,cardiac drugs such as organic nitrates, calcium channel blockers,sympatholytic agents, vasodilators, β-adrenergic receptor agonists andantagonists, α-adrenergic receptor agonists and antagonists, cardiacglycosides, anti-arrhythmic drugs, agents that affecthyperlipoproteinemias such as 3-hydroxymethylglutaryl-coenzyme A(HMG-CoA) inhibitors, anti-neoplastic agents such as alkylating agents,antimetabolites, natural products, antibiotics, and other drugs,immunomodulators, anti-diabetic agents, and anti-microbial agents suchas antibacterial agents, antiviral agents, antifungal agents,antiprotozoal agents, and antihelminthic agents.
 4. The method of claim1, wherein the enzyme is selected from the group consisting ofcytochrome P450, alkaline phosphatase, α-galactosidase, β-galactosidase,α-glucosidase, β-glucosidase, α-glucuronidase, β-glucuronidase,α-amylase, NADPH-cytochrome P450 reductase, cytochrome b₅,N-demethylase, O-demethylase, acetylcholinesterase,pseudocholinesterase, epoxide hydrolase, amidases, uridine diphosphate(UDP)-glucuronosyltransferases, phenol sulfotransferase, alcoholsulfotransferase, sterid sulfotransferase, and arylaminesulfotransferase, UDP-glycosyltransferases, purinephosphoribosyltransferase, N-acetyltransferases, glutathioneS-transferase, phenylethanolamine N-methyltransferase, non-specificN-methyltransferase, imidazole N-methyltransferase,catechol-O-methyltransferase, hydroxyindole-O-methyltransferase,S-methyltransferase, alcohol dehydrogenase, aldehyde dehydrogenase,xanthine oxidase, monoamine oxidases, diamine oxidases, flavoproteinN-oxidases, hydroxylases, aromatases, cysteine conjugate β-lyase, andalkylhydrazine oxidase.
 5. The method of claim 1, wherein the enzyme isendogenously expressed in the tissue.
 6. The method of claim 1, whereinthe enzyme has normal enzymatic activity.
 7. The method of claim 1,wherein the enzyme contains a polymorphism or mutation.
 8. The method ofclaim 1, wherein the enzyme is a recombinant enzyme.
 9. The method ofclaim 1, wherein the enzyme is cytochrome P450.
 10. The method of claim1, wherein the one or more metabolites are detected by liquidchromatography, mass spectrometry, nuclear magnetic resonance, orspectrophotometry. 11-22. (canceled)
 23. A method for determiningefficacy of a test agent, wherein efficacy comprises activity sufficientto decrease or eliminate hepatitis C virus in liver tissue comprising:A) incubating a test agent within a three-dimensional liver tissueengineered system; and B) measuring levels of hepatitis C virus.
 24. Amethod for determining efficacy of a test agent, wherein efficacycomprises activity sufficient to decrease or eliminate hepatitis C virusin liver tissue comprising: A) incubating a test agent within athree-dimensional liver tissue engineered system; and B) detectingimproved liver function.
 25. The method of claim 24, wherein improvedliver function is indicated by detecting normal enzyme levels, histologyor protein production and combinations thereof.
 26. The method of claim24, wherein the enzyme is SGOT, ALT or LDH.